Compositions of phosphorylated tau peptides and uses thereof

Abstract

To provide novel means capable of preventing neurodegenerative diseases such as Alzheimer's disease.SOLUTION: A liposome is provided comprising: a. a tau peptide; and b. a helper T cell epitope, the tau peptide being displayed on a surface of the liposome.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention is in the field of medicine. The present invention relates in particular to the liposome or co-administration of tau peptides. and methods for preventing or treating tauopathies such as Alzheimer's disease Regarding its use. [Background technology]

[0002] Alzheimer's disease (AD) is a progressive, debilitating disease that affects an estimated 44 million people worldwide. It is a neurodegenerative disease (Alzheimers.net). Currently available AD treatments in the clinic are clinically Aims to slow the progression of symptoms but does not target the pathogenic processes underlying the disease Unfortunately, these treatments are only minimally effective, and therefore additional preventative measures are not recommended. And there is an urgent need to develop and test therapeutic approaches.

[0003] A characteristic pathology of Alzheimer's disease is the formation of cells containing aggregated amyloid beta protein. accumulation of outer lytic plaques and intracellular "tangles" or aggregates of hyperphosphorylated tau protein The molecular events leading to the accumulation of these proteins are poorly characterized. In amyloids, abnormal cleavage of the amyloid precursor protein results in the formation of a protein that is capable of aggregating, including amino acids 1-42. It is hypothesized that this results in the accumulation of fragments that tend to bind to tau. It has been hypothesized that dysregulation of either or both of these enzymes leads to abnormal tau phosphorylation. After being hyperphosphorylated, tau loses its ability to effectively bind and stabilize microtubules. Instead, unbound and hyperphosphorylated tau accumulates in the cytoplasm of affected neurons. It is believed that the initial oligomers and then higher order aggregates are formed, the presence of which may be Raku negatively impacts the function of the neurons it forms through disruption of normal axonal transport. It is estimated that

[0004] In developed countries, individuals diagnosed with Alzheimer's disease or other dementia tauopathies Typically, cholinesterase inhibitors (e.g., Aricept®) or meman These drugs are used to treat It is well tolerated but has very modest efficacy. For example, Aricept (registered ®) delays the progression of symptoms by 6 to 12 months in approximately 50% of treated individuals. The treatment is non-pharmacological and focuses on the cognitive decline of patients and their ability to perform daily tasks. The focus is on enabling management.

[0005] Several published studies (Asuni AA et al., J Neurosci. 2007 Aug 22;27(34):9115 -29, Theunis C et al., PLoS One. 2013; 8(8): e72301, Kontsekova E et al., Alzhei Mers Res Ther. 2014 Aug 1;6(4):44) reported that an active vaccine containing tau peptides was effective in preventing the In rodent brains, which can induce anti-tau immune responses in mice or rats and in animals with Alzheimer's disease. It has been demonstrated that it can slow the rate of cognitive decline in models of pathology. An active vaccine against the biological tau protein has been shown to be effective in human patients with Alzheimer's disease. It has been shown to be immunogenic (Novak P et al., Lancet Neurology 2017, 16:12 3-134). WO 2010 / 115843 brochure describes the main protein tau. Antigenic phosphopeptides mimicking pathological phosphoepitopes and Alzheimer's disease and related compositions for therapeutic and diagnostic use in the treatment of tauopathies, including However, there are currently no effective vaccines approved to prevent the development of tau-mediated diseases. There are still no drugs on the market that can interrupt or slow the progression of the disease after it has started. There are no effective drugs on the market to prevent these diseases. There is an urgent need to identify new preventative measures (e.g., vaccines) that can prevent the disease. Summary of the Invention

[0006] In one general aspect, the invention provides a method for producing a cellular membrane comprising: a. a tau peptide, preferably a tau phosphopeptide; b. Helper T cell epitopes and wherein the tau peptide is displayed on the surface of the liposome.

[0007] In one embodiment, the liposome comprises at least one azido-containing toll-like receptor ligand. Preferably, the liposome further comprises a toll-like receptor 4 ligand and a toll-like receptor 4 ligand. The antibody further comprises at least one of the receptor-like receptor 9 ligands.

[0008] In a preferred embodiment, the present invention comprises: a. a tau peptide, preferably a tau phosphopeptide; b. helper T cell epitopes; ci Toll-like receptor 9 ligand, preferably a lipidated CpG oligonucleotide; and ii. Toll-like receptor 4 ligands, preferably Toll-like receptor 4 agonists with at least one of wherein the tau peptide is displayed on the surface of the liposome.

[0009] In a further preferred embodiment, the present invention provides a method for producing a composition comprising: a. tau phosphopeptide; b. helper T cell epitopes; c. lipidated CpG oligonucleotides; d. an adjuvant containing a Toll-like receptor 4 ligand; Including, The present invention relates to liposomes in which tau phosphopeptides are displayed on the surface of the liposomes.

[0010] In another general aspect, the invention provides a method for treating a tau peptide, preferably a tau phosphopeptide, comprising: and an immunogenic carrier conjugated thereto, wherein the tau peptide is carried via a linker. The linker is a polyethylene glycol. Cholesterol (PEG), succinimidyl 3-(bromoacetamido)propionate (SB AP), and m-maleimidobenzoyl-N-hydroxysuccinimide ester (M Immunogenic carriers useful in the present invention may include one or more of the following: Examples include, but are not limited to, keyhole limpet hemocyanin (KLH) , tetanus toxoid (TT), CRM197, and N. meningitidis-derived The outer membrane protein mixture (OMP), or derivatives thereof, is included.

[0011] In one preferred embodiment, the present invention provides a compound having the structure of formula (I):

[0012] [ka] or a structure of formula (II):

[0013] [ka] [In the formula, x is an integer from 0 to 10, preferably from 2 to 6, and most preferably 3; n is an integer of 2 to 11, preferably 3 to 11. The present invention relates to a conjugate having the formula:

[0014] A further aspect of the invention is a method for producing a liposome or conjugate of the invention in a pharmaceutically acceptable carrier. and a carrier, a method for preparing the pharmaceutical composition, and a method for treating a patient requiring the same. Induction of an immune response against tau or treatment of a neurodegenerative disease or disorder in a subject or the use of a pharmaceutical composition in the prevention thereof.

[0015] In one embodiment, the present invention provides a method for inducing an immune response in a subject suffering from a neurodegenerative disorder. or a method for treating a neurodegenerative disease or disorder in a subject in need thereof. The method relates to administering to a subject a liposome of the present invention and a pharmaceutically acceptable carrier. or a pharmaceutical composition comprising the conjugate of the present invention and a pharmaceutically acceptable carrier. Preferably, the method comprises administering to the subject a pharmaceutical composition comprising Pharmaceutical compositions of the present invention for immunizing against a mammalian animal, and medicaments of the present invention for boosting immunization The method includes administering the composition.

[0016] Further aspects, features, and advantages of the present invention are set forth in the following detailed description and accompanying drawings. A better understanding will be obtained from reading the claims.

[0017] The foregoing summary and the following detailed description of preferred embodiments of the present application should be read in conjunction with the accompanying drawings. However, the present application is directed to these detailed embodiments shown in the drawings. It should be understood that the present invention is not limited to the embodiments. [Brief explanation of the drawings]

[0018] [Figure 1] Figure 1 illustrates a novel vaccine according to an embodiment of the present invention: a tau liposome according to an embodiment of the present invention (top) and a tau conjugate according to an embodiment of the present invention (bottom). [Figure 2] FIG. 2 illustrates that a vaccine comprising a liposome (second-generation liposome) according to one embodiment of the present invention containing an encapsulated helper T cell epitope (e.g., tetanus polypeptide (tet)) activates helper T cells. [Figure 3] FIG. 3 illustrates that a vaccine comprising a conjugate according to one embodiment of the present invention containing a non-self or immunogenic carrier protein activates helper T cells. [Figure 4] 4 shows that tau vaccines according to embodiments of the invention induce sustained, high titers of anti-phospho-tau antibodies in rhesus macaques. The geometric mean endpoint titers per group, measured by enzyme-linked immunosorbent assay (ELISA) over time, are higher for vaccines comprising liposomes according to one embodiment of the invention (Liposome Z) or conjugates according to one embodiment of the invention (Conjugate A) than for control liposomal vaccines without helper T cell epitopes. [Figure 5] FIG. 5 shows that serum from rhesus macaques immunized with liposomes according to one embodiment of the present invention (Liposome Z) binds to pathological tau structures in human AD brain sections (left panel) compared to healthy human brain sections (right panel). [Figure 6]FIG. 6 shows that serum from rhesus macaques immunized with a conjugate according to one embodiment of the invention (Conjugate A), formulated in a composition containing soluble CpG and alum hydroxide, binds to pathological tau structures in human AD brain sections (top row) compared to healthy human brain sections (bottom row). [Figure 7] Figure 7 shows the titers of anti-phosphorylated tau antibodies in rhesus macaques induced by liposomal vaccines according to embodiments of the present invention, liposomes X, Y, and Z, each of which contains encapsulated T cell epitope T50 and one or more adjuvants. Titers were measured by ELISA and are presented as endpoint titers over time in individual monkeys. Specifically, Figure 7A shows the titers of anti-phosphorylated tau antibodies induced by liposome X adjuvanted solely with the TLR4 ligand MPLA (3D-(6-acyl)PHAD®). Figure 7B shows the titers of anti-phosphorylated tau antibodies induced by liposome Y adjuvanted solely with the TLR9 ligand (lipidated CpG oligonucleotide). Figure 7C shows the titers of anti-phosphorylated tau antibodies induced by Liposome Z adjuvanted with a combination of the TLR4 ligand MPLA (3D-(6-acyl)PHAD®) and a TLR9 ligand (lipidated CpG oligonucleotide). The combination of the two adjuvants also demonstrates less variability in antibody titers induced between individual monkeys. Figure 7D shows the geometric mean antibody titers for the above immunization groups and a control liposomal vaccine with the TLR4 ligand MPLA but no T cell epitope, demonstrating that vaccines according to embodiments of the invention produce higher titers of anti-phosphorylated tau antibodies than the control liposomal vaccine. Titers were measured by ELISA and are expressed as the geometric mean endpoint titer per group over time with a confidence interval of + / - 95%. [Figure 8]8 shows that immunization with a liposome vaccine (e.g., liposome X, Y, or Z) or a conjugate vaccine (conjugate A) according to one embodiment of the present invention induces antibody IgG titers specific to enriched paired helical fibrils (ePHFs) isolated from postmortem brains of Alzheimer's disease patients. Antibody titers were measured by Meso Scale Discovery (MSD) technology and are expressed as individual monkey values ​​and geometric mean + / - 95% CI at day 50 after the first immunization. [Figure 9] FIG. 9 shows that immunization with a liposomal vaccine according to one embodiment of the present invention (Liposome Z), which contains a combination of encapsulated T50 and a TLR4 ligand (3D-(6-acyl)PHAD®) and a TLR9 ligand (lipidated CpG oligonucleotide) as adjuvants, induces antibodies that bind mostly to the N-terminus of the phosphorylated tau peptide of SEQ ID NO: 2 ( FIG. 9A ), whereas monkeys immunized with a conjugate vaccine according to one embodiment of the present invention (Conjugate A) produce IgG antibodies that bind mostly to the C-terminal portion of the peptide for both the phosphorylated peptide (left) and the non-phosphorylated peptide (right) ( FIG. 9B ). [Figure 10]10A and 10B show that vaccination with a liposomal vaccine according to one embodiment of the present invention (Liposome S), which contains encapsulated T cell epitope T50 and a TLR4 ligand (3D-(6-acyl)PHAD®) as an adjuvant, induces significantly higher antibody titers in mice than a control liposomal vaccine (Liposome R, which has the TLR4 ligand, 3D-(6-acyl)PHAD®, but not the T cell epitope T50) and a liposomal vaccine according to one embodiment of the present invention (Liposome T), which contains the surface T cell epitope T57 (dipalmitoylated T50) and a TLR4 ligand (3D-(6-acyl)PHAD®). Antibody titers were measured by ELISA at 21 days (FIG. 10A) and 35 days (FIG. 10B) after the first immunization and are presented as individual values ​​and geometric mean ± 95% CI per group. (**:p<0.01, ***:p<0.001). [Figure 11] Figure 11 shows that encapsulation of T cell peptides T48 or T52 into liposomes (liposome M or N, respectively) induces T cell responses specific to the encapsulated peptide in mice. T cell responses were assessed by IFN-γ (Figure 11A) and IL-4 (Figure 11B) ELISPOT. [Figure 12] Figure 12 shows that liposomal vaccines containing encapsulated T cell epitopes (liposome L) and tethered T cell epitopes (liposome O) each induced higher tau phosphopeptide-specific antibody titers than the control liposomal vaccine without T cell epitopes. Liposome L, Liposome O, and the control liposome each further contained MPLA as an adjuvant. [Figure 13]Figure 13 shows that tau conjugates (KLH-TAUVAC-p7.1 or KLH-TAUVAC-p22.1) induce robust Ab titers against TfH cells and tau peptides in wild-type mice. Specifically, Figure 13A illustrates that groups of adult female Balb / C mice (n=14 per group) were immunized a total of four times with 100 μg of adjuvanted KLH-tau conjugate vaccine (KLH-TAUVAC-p7.1 or KLH-TAUVAC-p22.1), active placebo vaccine (KLH+alum or Ribi), or inactive placebo (PBS) according to the indicated schedule. Four animals from each immunization group were sacrificed 7 days after the primary immunization, and lymph nodes draining the injection site were harvested. Figure 13B shows the geometric mean percent TfH in draining lymph nodes by immunization group (n = 4 mice / group, analyzed individually). All groups receiving active vaccine or placebo had measurable TfH. Furthermore, animals receiving vaccine KLH-TAUVAC-p7.1, KLH-TAUVAC-p22.1, or active placebo KLH + alum had significantly more TfH than animals given the inactive placebo (p = 0.0044 for KLH-TAUVAC-p7.1, p = 0.0482 for KLH-TAUVAC-p22.1, p = 0.0063 for KLH, using ANOVA test followed by Dunnett's adjustment for multiple comparisons). Figures 13C-H show the change in serum titers from baseline (day 0) at four time points (days 14, 28, 56, and 84) after immunization, along with 95% confidence intervals for group means (n=5-10 animals). Asterisks indicate time points at which KLH-TAUVAC-induced antibody responses were significantly higher than those induced by active placebo (p≦0.05, as determined using ANOVA followed by Tukey's adjustment for multiple comparisons). More specifically, Figure 13C shows binding titers for phosphorylated tau peptide p7.1; Figure 13D shows binding titers for phosphorylated tau peptide p22.1; and Figure 13E shows binding titers for non-phosphorylated tau peptide 7.1.Figure 13F shows the binding titers to non-phosphorylated tau peptide 22.1, and Figures 13G and H show the binding titers to the carrier protein KLH, respectively. [Figure 14] Figure 14 shows that serum from mice immunized with tau conjugates also bound to pathological tau structures from other tauopathies. Pooled serum (n = 6 mice) from each vaccination group 84 days after primary immunization was used to stain brain tissue from a case of frontotemporal dementia with a MAPT mutation (MAPT P301S, frontal cortex), Pick's disease (frontal cortex), progressive supranuclear palsy (PSP, caudate nucleus), and a case of primary age-related tauopathy (PART, hippocampus). Serum from animals receiving the active vaccine highlighted tau-associated structures typical of each tauopathy, while serum from animals immunized with an active placebo (KLH-alum or KLH-Ribi) or an inactive placebo (PBS) failed to stain any of these structures. For reference, immunostaining with AT8 in the corresponding region is shown; scale bar = 50 μm. [Figure 15]Figure 15 shows that vaccine-induced antibodies reduce aggregated tau in an accelerated tauopathy model. Specifically, Figure 15A shows that 3-month-old P301L transgenic mice (n=15 / group) received stereotactic injections of human ePHFs preincubated with purified IgG from mice immunized with either KLH-TAUVAC-p7.1+RIBI or active placebo KLH+RIBI. Two months after injection, all mice were sacrificed, and the amount of aggregated tau in the total and sarkosyl-insoluble fractions in the mice was determined. Figures 15B and 15C show the total (B) and sarkosyl-insoluble (C) fractions collected from the injected hemisphere of each animal. The graphs show the amount of tau measured by MSD. In both the total and insoluble fractions, brains of mice receiving ePHFs preincubated with IgG from mice immunized with KLH-TAUVAC-p7.1 had significantly less aggregated tau than mice receiving ePHFs preincubated with control antibody (p<0.0001, using ANOVA followed by Holm-Bonferroni adjustment for multiple comparisons). [Figure 16]Figure 16 shows that a tau conjugate according to one embodiment of the present invention (Conjugate B) induces high titers of antibodies against phosphorylated tau and ePHF in non-human primates. Rhesus macaques were immunized with alum and CpG-adjuvanted KLH-TAUVAC-p7.1 (n=6) or KLH (n=2) on days 1, 29, 85, and 169. Blood was collected every 14 days. Specifically, Figure 16A shows that sera from animals immunized with KLH-TAUVAC-p7.1 were tested for reactivity to the immunizing peptide p7.1 using ELISA. Figure 16B shows that sera collected from all animals 50 days after primary immunization had measurable antibody levels against human ePHF using MSD, with three of six animals showing high reactivity to this antigen. (Figure 16C) Serum collected from animals 50 days after primary immunization was applied to human brain sections from healthy individuals or AD patients. Post-immunization serum from the KLH-TAUVAC-p7.1 group stained pathological tau structures in AD brain tissue, i.e., neurofibrillary tangles, neuropil threads, and neuritic plaques, while serum from mice immunized with KLH failed to show any reactivity, and no staining was observed in control tissue. (Figure 16D) When tested in a tau immunodepletion assay, animals receiving KLH-TAUVAC-p7.1 possessed antibodies capable of binding and depleting tau species (p = 0.03 at day 50, using ANOVA followed by Dunnett's adjustment for multiple comparisons), whereas immunization with KLH did not elicit such antibodies. (Figure 16E) Pre- and post-immunization serum was also tested in a neutralization assay as serially diluted individual samples. Change from baseline (CFB) was calculated as the difference in FRET counts read between day -14 pre-vaccination (baseline) and each of days 50, 106, and 190 post-vaccination. The response at a particular post-vaccination day (day i) was then calculated as follows:Response = %FRET_day i - %FRET_baseline A general linear mixed model for response as described above, with animal as a random effect, was applied with vaccine group, day, and serum level variables and all their interactions treated as categorical variables. [Figure 17] Figure 17 shows that mice immunized with a conjugate vaccine (Conjugate A) according to one embodiment of the present invention and a combination of alum hydroxide (Alum) and oligo-CpG (CpG) adjuvant produce higher titer antibody responses to the vaccine peptide. Adult female C57BL / 6 mice (n=5-6 / group) were immunized intramuscularly with either 2 μg or 0.2 μg of Conjugate A vaccine, and the conjugate vaccine was administered either alone, with alum, with CpG, or with a combination of alum and CpG. All mice received a primary immunization on day 0 of the study, followed by a single booster immunization on day 28. The dose of the alum adjuvant was 500 μg / mouse / injection, and the dose of the CpG adjuvant was 20 μg / mouse / injection. The graph shows the results of a binding ELISA using serum collected from mice immunized with vaccine peptide T3.5 as the coating antigen. Mean T3.5-specific endpoint titers per group are plotted before immunization (day 0) and at two time points after immunization (days 28 and 42), with error bars representing standard error. The table shows statistical analysis of the results, comparing antibody titers using a nonparametric Kruskal-Wallis test and evaluating pairwise group comparisons using a Wilcoxon signed-rank test as a post-hoc Kruskal-Wallis test. Specifically, Figure 17A shows mice immunized with 2 μg of conjugate A vaccine. Figure 17B shows mice immunized with 0.2 μg of conjugate A vaccine. [Figure 18] FIG. 18 shows that tau vaccines according to embodiments of the invention with various ratios of tau peptides to T cell epitopes induce long-lasting, high-titer anti-phospho-tau antibodies in rhesus macaques. DETAILED DESCRIPTION OF THE INVENTION

[0019] Various publications, articles, and patents are cited or noted in the background and throughout the specification. Each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is expressly incorporated by reference in its entirety. Such discussion is intended to provide a context for the invention. The prior art, in whole or in part, is not related to any invention disclosed or claimed. This does not authorize the formation of a division.

[0020] Unless otherwise defined, all technical and scientific terms used herein are It has the same meaning as commonly understood by a person skilled in the art to which this invention pertains. Wherever possible, certain terms used herein have the meanings set forth herein.

[0021] As used in this specification and the appended claims, the singular forms "a" and "one" are used interchangeably. "(an)" and "that (th)" are used unless the context clearly indicates otherwise. It should be noted that plural referents are included unless indicated otherwise.

[0022] Unless otherwise stated, any numerical value, such as a concentration or concentration range, described herein is All such statements are to be understood as being modified in all instances by the term "about." Therefore, values ​​typically include ±10% of the stated value. For example, 1 mg / mL concentrations include 0.9mg / mL to 1.1mg / mL. Similarly, 1% to 10% The concentration range of (w / v) includes 0.9% (w / v) to 11% (w / v). The use of numerical ranges within the specification is expressly prohibited unless the context clearly dictates otherwise. All possible subranges, all individual numbers within that range (such ranges Includes integers within ), and explicitly includes fractions of values.

[0023] Unless otherwise specified, the term "at least" preceding a series of elements means that all elements in the series It is understood that the term "component" refers to all elements present in the invention as described herein. It will be apparent to those skilled in the art that many equivalents to the specific embodiments of the present invention may be realized or used no more than routine experimentation. Such equivalents are not intended to be encompassed by this invention. Figure.

[0024] As used herein, the terms "including," "including," "including" and "included" , "having," "having," "containing," or "containing"; Any other variation implies the inclusion of the stated integer or group of integers, but any other integer or It is understood that the term does not imply the exclusion of integers and is intended to be non-exclusive or open-ended. For example, compositions, mixtures, processes, methods, articles containing lists of elements The term "apparatus" or "apparatus" is not necessarily limited to only those elements not expressly listed or or any other matter not inherent in such composition, mixture, process, method, article, or device. Furthermore, unless expressly stated to the contrary, "Or" means an inclusive or, not an exclusive or. For example, if Condition A or B is satisfied by any one of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true is (or exists), and A and B are both true (or exist).

[0025] Those skilled in the art will appreciate that no particular reference to dimensions or characteristics of the components of the preferred invention will be made. As used herein, the terms "about," "approximately," "generally," "substantially," and "various" are used in conjunction with the following: and similar terms are used to indicate that the dimensions / characteristics described are not strict boundaries or parameters but are functional. It is understood that this indicates that the product is essentially the same or similar, and does not exclude minor variations therefrom. At a minimum, such references including numerical parameters should be construed as indicative of the degree of specificity that is understood by those skilled in the art. Accepted mathematical and engineering principles (e.g., rounding, measurement error or other systematic errors) , manufacturing tolerances, etc.) will be included that do not change the least significant figure.

[0026] Microtubule-associated protein tau, MAPT, neurofibrillary tangle protein, paired helical microtubules Also known as fibrillar-tau, PHF-tau, MAPTL, MTBT1, and the like, as used herein The term "tau" or "tau protein" refers to an abundant tau protein with multiple isoforms. Refers to proteins in the central and peripheral nervous systems. In the human central nervous system (CNS), selective splicing is Lysing results in six major fragments ranging in size from 352 to 441 amino acids in length. There are several tau isoforms (Hanger et al., Trends Mol Med. 15:112-9, 2009) Examples of tau include, but are not limited to, tau with four repeats and two insertions: The longest tau isoform (4R2N) of 441 amino acids, as well as three repeats The shortest (fetal) isoform (3R) is 352 amino acids long and has no insertion. Examples of tau include tau isoforms in the CNS, such as 300 "Big tau" expressed in peripheral nerves contains an additional residue (exon 4a) g tau) isoforms. Friedhoff et al., Biochimica et Biophysica Ac ta 1502 (2000) 122-132. Examples of tau include mRNA transcripts of 6762 nucleotides in length. The length of the sequence encoded by the transcript (NM_016835.4) is 758 amino acids. The protein human big tau or its isoforms are included. The amino acid sequence of Tobig-tau is represented in GenBank accession number NP_058519.3 As used herein, the term "tau" includes the tau protein found in cynomolgus monkeys (Macaca fascicularis). non-human species such as the Japanese tiger cricket (Pan troglodytes) or the Japanese chimpanzee (Pan troglodytes) As used herein, the term "tau" includes full-length wild-type tau homologs from Tau mutations, such as point mutations, fragments, insertions, deletions, and splice variants, The term "tau" also includes post-translational modifications of the tau amino acid sequence. Post-translational modifications include, but are not limited to, phosphorylation.

[0027] As used herein, the term "peptide" or "polypeptide" refers to a molecule that is bound to a peptide bond. Polymers composed of amino acid residues linked by related naturally occurring structures The term refers to synthetic variants of any size and non-naturally occurring analogues thereof. A peptide is a peptide of any desired structure, structure, or function. Typically, a peptide is a peptide of at least three amino acids. Peptides may be naturally occurring, recombinant, or synthetic, or any combination thereof. The synthetic peptides can be prepared, for example, by automated polypeptide synthesizers. Examples of tau peptides include those containing about 5 to about 30 amino acids. The length is preferably about 10 to about 25 amino acids, more preferably about 16 to about 21 amino acids. In the present disclosure, peptides include any peptide of tau protein having a length of 1 to 20 amino acids. List amino acids from N to C terminus using standard three-letter or one-letter amino acid abbreviations, with phosphate residues The group is indicated by "p". Examples of tau peptides useful in the present invention include, but are not limited to: a tau peptide containing any of the amino acid sequences of SEQ ID NOs: 1 to 12, or Identifies at least 75%, 80%, 85%, 90%, or more of any of the amino acid sequences of Nos. 1 to 12 or tau peptides having amino acid sequences that are 95% identical.

[0028] As used herein, the term "phosphopeptide" or "phosphoepitope" refers to a It also refers to a peptide phosphorylated at multiple amino acid residues. Examples of tau phosphopeptides include includes any tau peptide containing one or more phosphorylated amino acid residues Examples of tau phosphopeptides useful in the present invention include, but are not limited to, the following: A tau phosphopeptide containing any one of the amino acid sequences of SEQ ID NOs: 1 to 3 or 5 to 12, or or a sequence identical to any one of the amino acid sequences of SEQ ID NOs: 1 to 3 or 5 to 12, with at least 75%, 8 Tau phosphopeptides with amino acid sequences that are 0%, 85%, 90%, or 95% identical Contains chid.

[0029] The tau peptides of the present invention can be synthesized by solid phase peptide synthesis or recombinant expression systems. The automated peptide synthesizer is manufactured by Applied Biosystems (California). It is commercially available from a number of suppliers, including the University of California, San Diego, California. Systems include bacteria such as E. coli, yeast, insect cells, or mammalian cells. Procedures for recombinant expression are described in Sambrook et al., Molecular Cloning: A Laboratory Manual (CSHP Press, NY 2d ed., 1989).

[0030] Tau is a human "self" protein. This means that, in principle, there is no specific receptor for tau. All lymphocytes carrying the IL-1 receptor are lost during development (central tolerance) or peripherally This means that tolerance mechanisms must have made the patient refractory to the drug. in the development of vaccines against "altered self" proteins (e.g., tumor antigens). It has proven to be a significant obstacle.

[0031] The production of high-quality antibodies against antigens (self or infectious) is dependent on antibody-producing B lymphocytes. as well as CD4 + It also requires the action of T "helper" lymphocytes. CD4 + T cells are heavy provides major survival and maturation signals to B lymphocytes, and CD4 + T cell-deficient animals are immunosuppressed The CD4 + T cells are susceptible to tolerance mechanisms and are potent anti-self ( A further obstacle to generating an antibody response (e.g., anti-tau) is the lack of tau-reactive CD4 + T cells It is likely that they are rare or absent in the human / animal repertoire.

[0032] Without wishing to be bound by theory and in any way limiting the scope of the invention, this It is believed that this problem is circumvented by the vaccine compositions of the present invention.

[0033] In one embodiment, most or all of the HLA DR (human leukocyte antigen-antigen D related) molecules Liposomes containing tau peptides (including T cell epitopes capable of binding to all An example is shown at the top of Figure 1). T cell epitopes are then expressed as CD4 + Activating T cells It can activate tau-specific B cells and provide essential maturation and survival signals to tau-specific B cells ( In another embodiment, a conjugate of a tau peptide with a carrier protein is produced. (An example is shown at the bottom of Figure 1), which generates a strong helper T cell response (Figure 3). In embodiments, carrier-specific T cells provide survival and maturation signals to autoreactive B cells. Thus, tau-specific B cells undergo affinity maturation, immune response, and A critical signal for initiating globulin class switching and establishing long-term memory pools. Tau liposomes and tau conjugates can be used to deliver homologous or heterologous tau. In the immunization scheme, liposomes or Using either of the conjugates, high-quality antibodies against the tau antigen can be generated. Cut.

[0034] Liposomes In one general aspect, the invention provides a method for producing a cellular membrane comprising: a. a tau peptide, preferably a tau phosphopeptide; b. helper T cell epitopes; wherein the tau peptide is displayed on the surface of the liposome.

[0035] Liposomes according to embodiments of the present invention are referred to herein as "improved liposomes" or "improved liposomes." "liposome vaccine" or "liposome vaccine according to an embodiment of the present invention" or "Tauri" Also called "liposomes" or "optimized liposomal vaccines" or "second-generation liposomes" can be.

[0036] As used herein, the term "liposome" generally refers to a material with a high lipid content. These vesicles are made of lipids such as phospholipids and cholesterol. Lipids are generally organized in the form of lipid bilayers. Interspersed among the multiple onion-like shells of the layer are multilamellar lipid vesicles (MLVs) or encapsulates a volume contained within an amorphous central cavity. Luminous lipid vesicles are unilamellar lipid vesicles, i.e., they have a single peripheral bilayer surrounding a cavity. Large unilamellar vesicles (LUVs) are generally 100 nm to several microns in size. diameters of 100-200 nm or more, while small unilamellar lipid vesicles ( SUVs are generally less than 100 nm, for example, 20-100 nm, typically 15-3 It has a diameter of 0 mm.

[0037] According to certain embodiments, the liposomes comprise one or more tau peptides. According to embodiments, the tau peptides in the liposomes can be the same or different.

[0038] Any suitable tau peptide known to one of skill in the art may be used in the present invention in light of this disclosure. According to certain embodiments, one or more of the tau peptides may be In another embodiment, the tau peptide comprises the amino acid sequence of one of SEQ ID NOs: 1 to 12. One or more of the sequences have at least one amino acid sequence selected from SEQ ID NOs: 1 to 12. and the amino acid sequence is 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence of the target gene. wherein none of the amino acid residues are phosphorylated, or one or more amino acid residues are phosphorylated. The residue is phosphorylated.

[0039] According to certain embodiments, one or more of the tau peptides is a tau phosphopeptide. According to certain embodiments, the one or more tau phosphopeptides are selected from the group consisting of SEQ ID NO: 1 1 to 3 or 5 to 12, or one of the amino acid sequences of SEQ ID NOs: 1 to 3 or 5 to 12 amino acid sequences and at least 75%, 80%, 85%, 90%, or contains amino acid sequences that are 95% identical, where one or more of the amino acid residues shown Preferably, the tau phosphopeptide is selected from the group consisting of SEQ ID NOs: 1 to 3. The tau peptide may be amidated at the C-terminus.

[0040] According to an embodiment of the present application, the tau peptide is displayed on the surface of the liposome. The tau peptide, preferably a tau phosphopeptide, can be prepared by any method known in the art in light of the present disclosure. The contents of the liposomes can be displayed on the surface using methods such as Nos. 8,647,631 and 8,647,631, which are incorporated herein by reference. See the related disclosure in US Pat. No. 9,687,447. According to certain embodiments, One or more tau peptides, including a phosphopeptide, are attached to the surface of the liposome. and one or more modifications, such as palmitoylation or dodecylation, that allow the antibody to be presented on the surface. The amino acid further comprises multiple modifications. To facilitate modification, Lys, Cys, or optionally This can be achieved by adding additional amino acid residues such as Ser or Thr to the tau peptide. The position of the lipid anchor can induce various conformations of the peptide sequence. has been reported (Hickman et al., J. Biol. Chem. vol. 286, NO. 16, pp. 13966-1399 76, April 22, 2011). Without wishing to be bound by theory, adding hydrophobic moieties to both ends It is thought that increasing the pathological beta-sheet conformation of tau peptides may increase the Therefore, one or more tau peptides may contain hydrophobic moieties at both ends. The modified tau peptide may be amidated at the C-terminus. The tau peptide displayed on the surface of the mouse is one of SEQ ID NOs: 27 to 38. It consists of a sequence of amino acids.

[0041] As used herein, the term "helper T cell epitope" refers to a molecule that binds to a helper T cell. A helper T cell epitope is a polypeptide containing an epitope that can be recognized by a helper T cell. Examples of helper T cell epitopes include including, but not limited to, tetanus toxoid (e.g., P2 and P30 epitopes) T2 and T30, respectively), hepatitis B surface antigen, cholera toxin B, toxoid, diphtheria toxoid, measles virus F protein, Chlamydia trachomatis ( Chlamydia trachomatis major outer membrane protein, Plasmodium falcip arum) circumsporozite T, P. falciparum CS antigen, Schistosoma mansoni triosephosphate isomerase, Bordetella pertussis, Clostridium tetani, Pertusaria Pertusaria trachythallina, Escherichia coli TraT, and influenza virus hemagglutinin (HA).

[0042] Any suitable helper T cell epitope known to one of skill in the art may be used in the present invention in light of this disclosure. According to a particular embodiment, helper T cell epitopes can be used in At least one amino acid sequence selected from the group consisting of SEQ ID NO: 23 to SEQ ID NO: 26 Preferably, the helper T cell epitope comprises one or more amino acids, e.g. For example, Val(V), Ala(A), Arg(R), Gly(G), Ser(S), Ly SEQ ID NO:2 fused together via a linker, such as a peptide linker comprising s(K) The linker may include two or more of the amino acid sequences of SEQ ID NO:3 to SEQ ID NO:26. Preferably, it is 1 to 5 amino acids. The fragment contains one or more linkers selected from the group consisting of VVR, GS, RR, and RK. Three or more of the amino acid sequences of SEQ ID NO: 23 to SEQ ID NO: 26 fused together via The helper T cell epitope may be amidated at its C-terminus.

[0043] According to an embodiment of the present application, the helper T cell epitope may be, for example, a covalently bound hydrophobic The liposome may be tethered by a functional moiety and incorporated onto the surface of the liposome, The hydrophobic moiety can be alkyl groups, fatty acids, triglycerides, diglycerides, steroids, sulfonates, etc. an ingolipid, glycolipid, or phospholipid, in particular an alkyl or fatty acid, in particular at least 3 carbon atoms, in particular at least 4 carbon atoms, in particular at least 6 carbon atoms, in particular at least At least 8 carbon atoms, particularly at least 12 carbon atoms, especially at least 16 carbon atoms In one embodiment of the present invention, the hydrophobic moiety is a palmitic Alternatively, the helper T cell epitope can be encapsulated in a liposome. According to certain embodiments, the helper T cell epitopes can be incorporated into the liposome. It is enclosed in a capsule.

[0044] Helper T cell epitopes can be identified using methods known in the art in light of the present disclosure. The desired location in the liposome can be modified. A useful helper T cell epitope in this context is one of SEQ ID NO: 39 to SEQ ID NO: 44. Preferably, the helper T cell epitope comprises one amino acid sequence selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 14. It consists of an amino acid sequence selected from the group consisting of sequence no. 17.

[0045] According to certain embodiments, the liposomes contain a tau peptide and a helper T cell epitope. in a weight ratio of 1:1, 2:1, 3:1, 4:1, 5:1, or 6:1.

[0046] In one embodiment, the liposome comprises at least one azido-containing toll-like receptor ligand. Thus, in another general aspect, the present invention provides a method for treating a pulmonary arthritis, comprising: a. a tau peptide, preferably a tau phosphopeptide; b. helper T cell epitopes; ci Toll-like receptor 9 ligand, and ii. Toll-like receptor 4 ligand with at least one of The present invention relates to a liposome comprising:

[0047] As used herein, the term "Toll-like receptor" or "TLR" refers to a receptor that is involved in the innate immune response. It refers to a class of pattern recognition receptor (PRR) proteins that play a major role in T LR is a pathogen-associated molecule from microbial pathogens such as bacteria, fungi, parasites, and viruses. TLRs recognize antigen-modifying antigens (PAMPs), which can be distinguished from host molecules. , a transmembrane protein that typically functions as a dimer and is expressed in antigen-presenting dendritic cells and phagocytic It is expressed by cells involved in the innate immune response, including effector macrophages. The ten human TLR family members, namely TLR1 to TLR10, as well as At least 12 murine TLR family members, namely TLR1-TLR9 and There are TLR11 to TLR13, and they recognize different types of antigens. For example, TLR4 binds to lipopolysaccharide (LPS), a component present in many Gram-negative bacteria. ), as well as low-density lipoproteins, beta-defensins, and heat shock proteins TLR9 recognizes viral proteins, polysaccharides, and endogenous proteins such as proteins. Unmethylated cytosine-phosphorylamine, abundant in prokaryotic genomes but rare in vertebrate genomes Nucleotide, activated by carboxyl-guanine (CpG) single- or double-stranded dinucleotides TLR activation is mediated by type I interferon (IFN), inflammation, and A series of cytokines and chemokines are produced, leading to the induction of immune responses. Ultimately, this inflammation also activates the adaptive immune system, which then leads to inflammation. This results in the elimination of pathogens and infected cells.

[0048] As used herein, the term "ligand" refers to a molecule that binds to a biological molecule (e.g., a receptor) and forms a complex with the molecule. According to certain embodiments, the toll-like receptor ligand Gand is a toll-like receptor agonist.

[0049] As used herein, the term "agent" refers to a substance that binds to one or more TLRs and binds to the receptor. Agonists are molecules that induce a receptor-mediated response. For example, agonists induce, stimulate, or inhibit the activity of a receptor. Such activity can be increased, activated, promoted, enhanced, or upregulated. For example, a TLR4 or TLR9 agonist may induce a vasoconstriction on the bound receptor. The agent can activate or increase cell signaling via Binds to, but is not limited to, nucleic acids, small molecules, proteins, carbohydrates, lipids, or receptors or any other molecule that interacts with the activity of the natural receptor ligand. The agent can mimic the structure of the receptor so that it can be recognized by the receptor. Homologous to these natural receptor ligands in terms of sequence, conformation, charge, or other characteristics This recognition allows the cell to recognize the receptor in the same way as if the natural receptor ligand were present. Physiological and / or biochemical changes within cells that respond to the presence of an agent in the same manner According to certain embodiments, the Toll-like receptor agonist is a Toll The receptor is at least one of a toll-like receptor 4 agonist and a toll-like receptor 9 agonist.

[0050] As used herein, the term "Toll-like receptor 4 agonist" refers to a compound that acts as an agonist of TLR4. The term "toll-like receptor 4 receptor antagonist" refers to any compound that acts as a toll-like receptor 4 receptor antagonist known to those of skill in the art. Agents useful in the present invention may be used in light of this disclosure. Examples of receptor-like receptor 4 ligands include, but are not limited to, monophosphoryl lipid A ( As used herein, the term "monophosphorylated leukocyte immunoglobulin (MPLA) is used to describe TLR4 agonists. "Methyl-methylenediamine-2-phosphate dehydrogenase (MPD)" or "MPLA" is a phospholipid that inhibits the biological activity of gram-negative bacterial lipopolysaccharide (LPS) endotoxins. MPLA is a modified form of lipid A, the active moiety of MPLA, which maintains its immunostimulatory activity. However, it is less toxic than LPS. As a vaccine adjuvant, MPLA is effective in immunizing vaccine antigens. Examples of MPLAs include, but are not limited to, Although not specified, 3-O-deacylated 4'-monophosphoryl lipid A, monophosphoryl hexa -acyl lipid A, 3-deacylated, monophosphoryl 3-deacylated lipid A, and structurally related MPLAs useful in the present invention include those known in the art. or Avanti Polar Lipids (Alabama, USA) labaster's 3D-(6-acyl) PHAD®, PHAD® ), PHAD®-504, 3D-PHAD®, or various commercial These compounds can be obtained from commercial sources such as the publicly available MPL™. Therefore, the toll-like receptor 4 agonist is MPLA. "TLR9 agonist" refers to any compound that acts as an agonist of TLR9. Any suitable Toll-like receptor 9 agonist known to the art may be used in the present invention in light of this disclosure. Examples of toll-like receptor 9 ligands useful in the present invention include those Examples of TLR9 agonists include, but are not limited to, CpG oligonucleotides.

[0051] As used herein, the terms "CpG oligonucleotide," "CpG oligodeoxynucleotide," "CpG ODN" refers to a nucleic acid sequence that contains at least one CpG motif. As used herein, "oligonucleotide" refers to an oligonucleotide. "Oxynucleotide," or "ODN," means a nucleic acid formed from multiple linked nucleotide units. Such oligonucleotides are polynucleotides that are synthesized from existing nucleic acid sources. As used herein, the term "aromatic" refers to a compound that can be obtained by a method known as a "romatic" or "aromatic" compound. The term "CpG motif" refers to a CpG motif that has a phosphate bond or a phosphodiester backbone or other Unmethylated cytosine-phosphate-guanine (CpG) linked by internucleotide bonds ) nucleotides containing a dinucleotide (i.e., cytosine (C) followed by guanine (G)) This refers to a dsDNA sequence.

[0052] According to a particular embodiment, the CpG oligonucleotide is lipidated, i.e. It is conjugated (covalently attached) to a lipid moiety.

[0053] As used herein, a "lipid moiety" refers to a moiety containing a lipophilic structure. groups, fatty acids, triglycerides, diglycerides, steroids, sphingolipids, glycolipids, or The lipid moiety, such as a phospholipid or sterol, especially cholesterol, or a fatty acid, is located in the nucleus. When attached to highly hydrophilic molecules such as acids, the binding of plasma proteins results in hydrophilic molecules Furthermore, the circulating half-life of certain proteins, such as lipoproteins, can be substantially increased. Binding to plasma proteins involves binding to corresponding lipoprotein receptors (e.g., LDL receptors, Uptake into specific tissues that express HDL receptors or scavenger receptors (SR-B1) In particular, phosphopeptides and / or CpG ions have been shown to increase The lipid moiety conjugated to the oligonucleotide may be This allows the oligonucleotide to be anchored within the membrane of the liposome via the hydrophobic portion.

[0054] According to certain embodiments, in view of the present disclosure, the CpG oligonucleotide may be any suitable It may include an internucleotide bond.

[0055] As used herein, the term "internucleotide linkage" refers to the linkage between adjacent nucleosides. It consists of a phosphate atom and a charged or neutral group, and connects two nucleotides together via their sugars. Examples of internucleotide bonds include phosphodiester (PO), phosphorothioate (P). thioate (ps), phosphorodithioate (ps2), methylphosphonate (mp), and methyl phosphorothioate (rp). Thioates, methylphosphonates, and methylphosphorothioates are stabilized nucleotides nucleotide linkage, while phosphodiester is a naturally occurring internucleotide linkage. Oligonucleotide phosphorothioates are typically Rp and Sp phosphorothioates. It is synthesized as a random racemic mixture of bonds.

[0056] Any suitable CpG oligonucleotide known to those of skill in the art may be used in the present invention in light of this disclosure. Examples of such CpG oligonucleotides include CpG2006 (also known as CpG7909), CpG 1018, CpG2395, CpG2216, or CpG2336.

[0057] CpG oligonucleotides may be liposomally linked using methods known in the art in light of this disclosure. In some embodiments, the 3' end of the CpG oligonucleotide can be , optionally via a PEG linker, and covalently linked to a cholesterol molecule via a phosphate bond. Other lipophilic moieties may also be covalently attached to the 3' end of the CpG oligonucleotide. For example, a CpG oligonucleotide can optionally contain a PEG linker. The lipid anchor, i.e., one palmitoyl group, is attached via the liposome to the phospholipid. carboxylic acid chain (using Pal-OH or similar, activated for coupling) or 2 Palmitic acid (e.g., 1,2-dipalmitoyl-sn-glycero-3-phosphoeta) Use N-(succinyl) or similar, activated for coupling ), the contents of which are incorporated herein by reference. See the related disclosure in U.S. Patent No. 7,741,297. The length can vary, for example, from 1 to 5 PEG units.

[0058] Other linkers also link CpG oligonucleotides to lipophilic moieties (such as cholesterol molecules). Examples include, but are not limited to, Contains alkyl spacers with 3 to 12 carbons. Suitable for oligonucleotide chemistry. In some embodiments, a short, covalent linker is required as the aminodiol. No linker is used for attachment. See, for example, Ries et al., “Convenient synthesis and application of versatile nucle ic acid lipid membrane anchors in the assembly and fusion of liposomes, Org. Bio See Mol. Chem., 2015, 13, 9673.

[0059] According to certain embodiments, lipidated CpG oligonucleotides useful in the present invention are nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 22, The nucleotide sequence contains one or more phosphorothioate internucleotide linkages. , and is covalently bound to at least one cholesterol via a linker. A linker is used to covalently attach the CpG oligonucleotide to a cholesterol molecule. Preferably, the linker comprises polyethylene glycol (PEG).

[0060] According to certain embodiments, the liposomes comprise: a. tau phosphopeptide; b. helper T cell epitopes; c. lipidated CpG oligonucleotides; d. Toll-like receptor 4 ligand and The tau phosphopeptide is displayed on the surface of the liposome, and the liposome is capable of inhibiting T helper cell proliferation. The pitope is encapsulated within the liposome.

[0061] According to certain embodiments, the liposomes comprise: a. Tau having an amino acid sequence selected from the group consisting of SEQ ID NO: 27 to SEQ ID NO: 38 A peptide, b. A herbicidal drug having an amino acid sequence selected from the group consisting of SEQ ID NO: 39 to SEQ ID NO: 44 per T cell epitope, preferably selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO: 17 a helper T cell epitope consisting of an amino acid sequence c. having a nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 22; containing one or more phosphorothioate internucleotide linkages and a lipidated CpG oligonucleotide covalently linked to at least one cholesterol; d. Monophosphoryl lipid A (MPLA) and Includes.

[0062] According to certain embodiments, the liposomes are 1,2-dimyristoyl-sn-glycero- 3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phospho from the group consisting of di-3'-rac-glycerol (DMPG), and cholesterol Further comprising one or more selected lipids.

[0063] According to certain embodiments, the liposomes further comprise a buffer. Any suitable buffer may be used in the present invention in light of this disclosure. In certain embodiments, the liposomes comprise phosphate buffered saline. Contains stigma and sucrose.

[0064] According to certain embodiments, the liposomes are composed of DMPC, DMPG, cholesterol, tau Phosphopeptides and helper T cell epitopes were mixed in the ratio of 9:1:7:0.07:0.04. Included in molar ratio.

[0065] The liposomes of the present invention are made using methods known in the art in light of this disclosure. It is possible.

[0066] An exemplary liposome of the present application is illustrated in Figure 1. More specifically, tau tetrapalmitoin The phosphopeptide (pTau peptide T3, SEQ ID NO: 28) was prepared by cleaving the phosphopeptide tau peptide T3 from the tau peptide T3. Lipid-attached C is presented on the surface of the liposome via two palmitic acid residues at the end of the molecule. TLR-9 ligands containing pG (adjuvant CpG7909-Chol) were covalently bound to The TLR-4 ligand is incorporated into the liposome membrane via the cholesterol that binds it. The adjuvant 3D-(6-acyl) PHAD® is also incorporated into the membrane. The helper T cell epitope (PAN-DR binding agent T50) is encapsulated. .

[0067] Conjugates In one general aspect, the invention provides tau peptides and immunogens conjugated thereto. and a hydroxyl group-containing carrier.

[0068] According to a particular embodiment, the conjugate has the following structure:

[0069] [ka] or a structure of formula (II):

[0070] [ka] [In the formula, x is an integer from 0 to 10, n is an integer of 2 to 15, preferably 3 to 11. It has.

[0071] According to certain embodiments, x is 1 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5 , 2 to 4, or an integer from 2 to 3. According to a particular embodiment, x is 3.

[0072] According to certain embodiments, n is 2 to 15, 3 to 11, 3 to 9, 3 to 8, or 3 to 7. is.

[0073] According to certain embodiments, the conjugate comprises one or more tau peptides. According to certain embodiments, the tau peptides of the conjugate may be the same or different.

[0074] According to certain embodiments, any suitable tau peptide may be used in the present invention in light of this disclosure. According to certain embodiments, one or more of the tau peptides can be used. The number is one of the amino acid sequences of SEQ ID NOs: 1 to 12, or At least 75%, 80%, 85%, 90%, or 95% identical to a single amino acid sequence wherein zero, one, or more of the amino acid residues are It is phosphorylated.

[0075] According to certain embodiments, one or more of the tau peptides is a tau phosphopeptide. According to certain embodiments, the one or more tau phosphopeptides are selected from the group consisting of SEQ ID NO: 1 1 to 3 or 5 to 12, or one of the amino acid sequences of SEQ ID NOs: 1 to 3 or 5 to 12 amino acid sequences and at least 75%, 80%, 85%, 90%, or contains amino acid sequences that are 95% identical, where one or more of the amino acid residues shown is multiply phosphorylated.

[0076] According to certain embodiments, the tau phosphopeptide has the amino acid sequence of one of SEQ ID NOs: 1-3. It consists of an acid sequence.

[0077] As used herein, the term "immunogenic carrier" refers to a compound that is coupled to a tau peptide. The immunogenic moiety coupled to the tau peptide is Induce an immune response, triggering the production of antibodies that can specifically bind to tau peptides An immunogenic portion is one that is recognized as foreign and therefore elicits an immune response from the host. Inducing, proteins, polypeptides, glycoproteins, complex polysaccharides, particles, nucleic acids, polynucleotides Any suitable immunogenic moiety known to those skilled in the art may be used, including but not limited to, a nucleotide. In view of the present disclosure, native carriers may be used in the present invention. For example, immunogenic carriers include keyhole limpet hemocyanin (KLH), tetanus toxoid, CRM197 (a non-toxic form of diphtheria toxin) from Neisseria meningitidis or a derivative thereof. , the immunogenic carrier is KLH or CRM197.

[0078] According to certain embodiments, the tau peptide is conjugated to a carrier via a linker. As used herein, the term "linker" refers to a molecule that connects an immunogenic carrier to a tau peptide. Any suitable linker known to those of skill in the art may be used in light of this disclosure. Linkers that can be used in the present invention include, for example, a single covalent bond, a substituted or unsubstituted linker. or unsubstituted alkyl, substituted or unsubstituted heteroalkyl moieties, polyethyleneglycol Poly(ethylene glycol) (PEG) linkers, peptide linkers, sugar-based linkers, and disulfide bonds are also available. or a cleavable linker such as a protease cleavage site, amino acids, or a combination thereof. Examples of linkers include polyethylene glycol (PEG), succinimides, Dimethyl 3-(bromoacetamido)propionate (SBAP), m-maleimidobenzoyl one or more of: Cys-N-hydroxysuccinimide ester (MBS); , Lys, or optionally one or more amino acids such as Ser or Thr , or a combination thereof.

[0079] According to certain embodiments, the linker is (C2H4O)x-cysteine-acetamide Propionamide or m-Maleimidobenzoyl-N-hydroxysuccinimide and ter-cysteine-(C2H4O)x, wherein x is an integer from 0 to 10, for example: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10].

[0080] According to certain embodiments, the carrier is covalently attached to the N-terminus of the tau peptide via a linker. are.

[0081] According to other particular embodiments, the carrier is covalently attached to the C-terminus of the tau peptide via a linker. It fits together.

[0082] According to certain embodiments, the conjugate has the following structure:

[0083] [ka] [In the formula, n is an integer of 2 to 15, preferably 3 to 11, and more preferably 3 to 7] It has.

[0084] The conjugates of the present invention can be made by methods known in the art in light of this disclosure. For example, the conjugate can be succinimidyl-3-(bromo Cetoamido)propionate (SBAP):

[0085] [ka] with the amino group of CRM197 to form an amide bond. This CRM197 precursor can then be released at its N-terminus or its C-terminus. Taupe conjugated with a PEG-cysteine ​​linker bearing a nucleophilic thiol group tau phosphopeptide by reacting with a phosphotau peptide (e.g., phosphorylated tau peptide of SEQ ID NO: 2) to produce a tau phosphopeptide. A doconjugate can be formed.

[0086] An exemplary conjugate according to one embodiment of the present application is illustrated in Figure 1. More specifically, Multiple tau phosphopeptides (pTau peptide T3.76) are expressed in the carrier protein CRM197 is covalently bonded to

[0087] Pharmaceutical Composition In one general aspect, the invention provides a therapeutically effective amount of a liposome or conjugate of the invention. and a pharmaceutical composition comprising the compound of formula (I) together with a pharmaceutically acceptable excipient and / or carrier. Pharmaceutically acceptable excipients and / or carriers are well known in the art (see Remington's P See Pharmaceutical Science (15th ed.), Mack Publishing Company, Easton, Pa., 1980. The preferred formulation of the pharmaceutical composition depends on the intended mode of administration and therapeutic application. The composition includes those commonly used to formulate pharmaceutical compositions for animal or human administration. It may contain a pharmaceutically acceptable, non-toxic carrier or diluent, defined as a vehicle. The diluent is selected so as not to affect the biological activity of the combination. Examples of diluents are distilled water, physiological phosphate-buffered saline, Ringer's solution, dextrose solution, and and Hank's solution. In addition, the pharmaceutical composition or formulation may contain other carriers, adjuvants, Carriers, excipients, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like may also be included. It will be understood that the characteristics of the formulation or diluent will depend on the route of administration of a particular application.

[0088] The pharmaceutical composition can contain a mixture of the same immunogenic tau peptides. The pharmaceutical composition may contain a mixture of different immunogenic tau peptides of the invention. .

[0089] Another problem associated with vaccines against neurological diseases is that they require exceptionally high levels of This is because the target antigen of the vaccine is located in the brain, and a high antibody titer may be required. The brain is protected from the blood by a special cellular structure called the blood-brain barrier (BBB). The BBB restricts the passage of substances from the circulation into the brain. It prevents toxins, microorganisms, etc. from entering the central nervous system. The BBB also functions as a barrier to immune mediators. It prevents substances (such as antibodies) from effectively entering the interstitial and cerebrospinal fluids that surround the brain. It also has potentially more undesirable effects, such as

[0090] Approximately 0.1% of antibodies present in the systemic circulation cross the BBB and enter the brain. Because systemic titers induced by vaccines targeting NS antigens are effective in the brain This means that the titer must be at least 1000 times higher than the minimum effective titer.

[0091] According to a particular embodiment, the pharmaceutical composition of the present invention therefore comprises one or more suitable anti-inflammatory drugs. Therefore, the present invention is not limited to the liposome or conjugate. The tau peptides of the invention can be administered in combination with a suitable adjuvant to achieve a desired immune response in a subject. Suitable adjuvants may be administered in combination with the liposomes or It can be administered before, after, or simultaneously with the administration of the or conjugate. A strong adjuvant induces conformational changes in the immunogen that affect the qualitative form of the response. They augment the natural response to immunogens without causing a reaction. Examples of adjuvants are aluminum hydroxide, aluminum phosphate, and aluminum sulfate Such adjuvants include the MPLA class (3-de-O-alum). Sylated Monophosphoryl Lipid A (MPL™), Monophosphoryl Hexa-acyl Lipid A 3-Deacylation Synthesis (3D-(6-Acyl) PHAD®), PHAD™, PHAD®-504, 3D-PHAD® (lipid A), polyglutamine Other specific immunostimulatory agents, such as polymeric or monomeric amino acids, such as phosphate or polylysine. Such adjuvants may be used with or without muramyl. Peptides (e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (t hr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (n or-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanyl Lanin-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryl) Oxy)-ethylamine (MTP-PE), N-acetylglucosaminyl-N-acetyl Muramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DT Other compounds, such as P-DPP (Dysfunctional Polysaccharides), Theramide™, or other bacterial cell wall components The oil-in-water emulsion may be used with or without specific immunostimulants such as: Contains 5% squalene, 0.5% Tween 80, and 0.5% Span 85. MF59 (optionally containing various amounts of MTP-PE) (using a microfluidizer) (See WO 90 / 14837) 0% squalene, 0.4% Tween 80, 5% Pluronic block polymer L 121, and SAF containing thr-MDP (microfluidized or or vortexing to produce emulsions with larger particle sizes), Ribi (commercial Targeted adjuvant system (RAS) (Ribi ImmunoChem, Hamburg, MT) 1 liter) 0.2% Tween 80, and monophosphoryl lipid A (MPL (trademark) )), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL™ + CWS (Detox™) Other adjuvants include complete Freund's adjuvant and several bacterial cell wall components. (CFA), and interleukins (IL-1, IL-2, and IL-12), Macrophage colony-stimulating factor (M-CSF) and tumor necrosis factor (TNF) Cytokines are included.

[0092] As used herein in the context of administering two or more treatments to a subject, the term "combination" refers to a combination of two or more treatments. "In combination" refers to the use of more than one therapeutic agent. There is no limitation on the order in which the first therapeutic agent (e.g., a compound described herein) is administered to a subject. The composition described above) may be administered to the subject at least 5 minutes, 15 minutes, 30 minutes, or 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, if or 12 weeks prior), simultaneously with, or subsequent to (e.g., 5, 15, 30 minutes Minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks later).

[0093] The pharmaceutical compositions of the present invention can be formulated by methods well known in the art. The optimum ratio of each component can be determined by techniques well known to those skilled in the art in light of this disclosure. It is possible.

[0094] How to use Another general aspect of the invention is to administer to a subject a pharmaceutical composition according to an embodiment of the invention. and detecting tau protein in a subject suffering from a neurodegenerative disease, disorder, or condition, including According to a particular embodiment, the immune response is a method for inducing an immune response against a phosphorylated protein. It is directed against a protein, preferably ePHF.

[0095] Another general aspect of the invention is to administer to a subject a neurodegenerative disease, disorder, or condition, a therapeutic agent according to the invention, or The present invention relates to a method of treating or preventing a disease comprising administering a pharmaceutical composition according to the embodiment.

[0096] As used herein, the terms "induce" and "stimulate" and variations thereof refer to the activation of cells. Induction of an immune response includes, for example, increasing immune cell populations. Activation, proliferation, or maturation of colonies, increased cytokine production, and / or increased immunity In certain embodiments, induction of an immune response may include: Increased proliferation of B cells, production of antigen-specific antibodies, increased proliferation of antigen-specific T cells, dendritic cells Improved antigen presentation and / or specific cytokines, chemokines, and costimulatory markers Increased expression of KAR can be included.

[0097] The ability to induce or stimulate an anti-tau immune response upon administration to an animal or human organism is currently unknown. in vitro or in vivo using a variety of assays that are standard in the field can be used to assess the initiation and activation of immune responses. For a general description of the available techniques, see, for example, Coligan et al. (1992 and 1994, Current Press, rotocols in Immunology; ed. J Wiley & Sons Inc, National Institute of Health) Cellular immunity can be measured by methods readily known in the art, for example, For example, CD4 + and CD8 + Activated effector cells, including those derived from T cells by measuring the cytokine profile secreted by the cells (e.g., ELISP Quantification of IL-4 or IFN-gamma producing cells by OT), activity of immune effector cells by determining the cytotoxicity state (e.g., classical [3H]thymidine incorporation) of T cell proliferation assay), by assaying antigen-specific T lymphocytes in sensitized subjects (e.g., peptide-specific lysis in cytotoxicity assays) can be performed.

[0098] The ability to stimulate a cellular and / or humoral response can be determined by administering to a subject a biological sample (e.g., blood, plasma, serum, PBMC, urine, saliva, feces, CSF, or lymph) into a pharmaceutical composition By testing for the presence of antibodies directed against the administered immunogenic tau peptide in the culture medium. (e.g., Harlow, 1989, Antibodies, Cold Spring Harbor) For example, the production of antibodies in response to administration of a composition providing an immunogen. Titers were determined by enzyme-linked immunosorbent assay (ELISA), dot blot, and SDS-PAGE. Measured by gel, ELISPOT, or antibody-dependent cellular phagocytosis (ADCP) assays It is possible.

[0099] As used herein, the term "subject" refers to an animal. According to certain embodiments, a subject non-primates (e.g., camels, donkeys, zebras, cattle, pigs, horses, goats, sheep, cat, dog, rat, rabbit, guinea pig, or mouse) or primate (e.g., In certain embodiments, the mammal is a mammal, including a monkey, a chimpanzee, or a human. , the subject is a human.

[0100] As used herein, the term "therapeutically effective amount" refers to an amount that achieves a desired biological or medical effect in a subject. A therapeutically effective amount refers to the amount of an active ingredient or component that elicits a therapeutic response. can be determined empirically and routinely. For example, the optimal In vitro assays can optionally be used to help identify a dose range. The selection of a particular effective dose depends on the disease to be treated or prevented, the symptoms involved, the weight of the patient, Consideration of several factors, including the patient's immune status, and other factors known to those skilled in the art. This can be determined by one skilled in the art (e.g., through clinical trials) based on: The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease, and should be decided by the judgment of the practitioner. The effective dose should be determined according to the individual patient's condition and the in vitro or may be extrapolated from dose-response curves derived from animal model test systems.

[0101] As used herein, the terms "treat," "treating," and "treatment" all refer to and at least one measurable physical parameter associated with a neurodegenerative disease, disorder, or condition. The term "remission or reversal of a disease" is intended to refer to a remission or reversal of a disease, which is not necessarily discernible in a subject. It need not be, but can be, discernible in a subject. "Treatment" and "treatment" refer to the act of causing the recurrence of a disease, disorder, or condition. It can also be said that the progression of the disease is prevented or at least slowed down. In certain embodiments, "treat," "treating," and "treatment" refer to the treatment of a neurodegenerative disease. Alleviating, preventing the occurrence or onset of one or more symptoms associated with a disease, disorder, or condition In certain embodiments, "treat" or "treating" refers to a shortening of the duration of the disease. and "treatment" refers to preventing the recurrence of a disease, disorder, or condition. "Treat," "treating," and "treatment" refer to the treatment of a disease, disorder, or condition. In certain embodiments, "treating" refers to increasing the survival of a subject with a disease. and "treatment" refers to the elimination of a disease, disorder, or condition in a subject.

[0102] According to certain embodiments, a therapeutically effective amount is one, two, three, four, or five of the following effects: (i) The amount of a therapeutic agent sufficient to achieve one or more of the following: (i) the disease being treated (ii) a reduction in the severity or amelioration of the disorder, condition, or symptoms associated therewith; a reduction in the duration of the disease, disorder, or condition being treated or symptoms associated therewith; ii) preventing the progression of the disease, disorder, or condition being treated or the symptoms associated therewith; iv) causing a recurrence of the disease, disorder, or condition being treated, or symptoms associated therewith; (v) preventing or reducing the occurrence of the disease, disorder, or condition being treated or symptoms associated therewith; (vi) the treatment of a disease, disorder, or condition, or symptoms associated therewith; (vii) preventing the recurrence of the disease, disorder, or condition being treated, or related thereto; (viii) reducing hospitalization of symptomatic subjects; and (ix) reducing the length of hospitalization of subjects with symptoms associated therewith; (x) increasing survival of a subject having a condition or symptom associated therewith; inhibiting or alleviating the disease, disorder, or condition being treated, or the symptoms associated therewith, and and / or (xi) enhancing or improving the prophylactic or therapeutic efficacy of another therapeutic agent.

[0103] As used herein, a "neurodegenerative disease, disorder, or condition" includes any neurodegenerative disease, disorder, or condition that is within the skill of the art in light of this disclosure. Neurodegenerative diseases, disorders, or conditions known to humans include: Examples of diseases, disorders, or conditions include tau-related diseases, disorders, or conditions known as tauopathies. which neurodegenerative diseases caused by or associated with the formation of neurofibrillary lesions According to certain embodiments, the neurodegenerative disease, disorder, or condition includes , including but not limited to Alzheimer's disease, Parkinson's disease, Creutzfeldt's disease -Jakob disease, dementia pugilistica, Down syndrome, Gerstmann-Straussler-Scheinka - disease, inclusion body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis Stratified sclerosis, Guam Parkinson-dementia complex, non-Guam movement disorder with neurofibrillary tangles Neuronal diseases, argyrophilic grain dementia, corticobasal degeneration, Lewy dementia, amyotrophic lateral sclerosis Sclerosis, diffuse neurofibrillary tangles with calcification, frontotemporal dementia, preferably Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), Frontotemporal dementia, Hallervorden-Spatz disease, multiple system atrophy, Niemann-Pick disease Type C, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, Tangle-only dementia, postencephalitic parkinsonism, myotonic dystrophy, chronic traumatic brain Disorders of the Tauocyte Epithelial Tissue (CTE), Primary Age-Related Tauopathy (PART), or Dementia with Lewy Bodies ( Any of the diseases or disorders that exhibit coexistence of tau and amyloid pathology, including LBD According to certain embodiments, the neurodegenerative disease, disorder, or condition is Alzheimer's disease. Having Heimer's disease or another tauopathy.

[0104] The present invention also provides a method for treating tau deficiency, comprising administering to a subject a therapeutically effective amount of tau deficiency inhibitor under conditions effective to promote clearance of tau aggregates from the brain of the subject, 2. A method for isolating tau from the brain of a subject, comprising administering to the subject a pharmaceutical composition according to one embodiment of the present invention. Also provided are methods for promoting the clearance of aggregates. According to certain embodiments, tau aggregates are neurogenic. fibrillary tangles or their pathological tau precursors.

[0105] The present invention also provides a method for delaying the progression of behavioral phenotypes associated with tau pathology in a subject. administering to a subject a pharmaceutical composition according to one embodiment of the present invention under conditions effective for Also provided are methods of slowing the progression of behavioral phenotypes associated with tau pathology in a subject.

[0106] In a preferred embodiment of the present invention, the pharmaceutical composition according to one embodiment of the present invention is administered Administration of tau peptides results in activity against tau peptides and pathological forms of tau in the subject. Induce a virulent immune response and thus facilitate the clearance of associated tau aggregates, potentially contributing to tau pathology. Slow the progression of associated behaviors and / or treat the underlying tauopathy. According to this aspect of the invention, the immune response is humoral (antibody-mediated) directed against tau peptides. mediated) responses and cells (antigen specific) directed against T cell epitopes or immunogenic carriers. This includes the beneficial generation of responses (mediated by allogeneic T cells or their secreted products).

[0107] As used herein, behavioral phenotypes associated with tau pathology include, but are not limited to, However, cognitive impairment, early personality changes and disinhibition, apathy, apraxia, mutism, apraxia, Persistence, stereotyped movements / behaviors, hyper-mouthiness, confusion, inability to plan or structure sequential tasks, selfishness / insensitivity Perception is impaired, with antisocial traits, lack of empathy, stammering, frequent erroneous errors, but comprehension is relatively preserved. agrammatic speech, impaired comprehension and lexical detection deficits, slowly progressive gait instability, Pacemaker, freezing, frequent falls, levodopa-refractory axial rigidity, supranuclear palsy, square wave eye movements movement, slow vertical saccades, pseudobulbar palsy, limb apraxia, dystonia, cortical sensory degeneration Symptoms include loss of consciousness, and tremors.

[0108] In practicing the methods of the invention, an immunogenic peptide or antibody of the invention is administered. Previously diagnosed with or at risk of developing Alzheimer's disease or other tauopathy Subjects with autism, tau aggregates in the brain, or tangle-associated behavioral phenotypes It is preferable to select subjects who are at risk for disease but have not yet developed the disease. This includes asymptomatic individuals and currently symptomatic patients. In this case, virtually everyone is at risk for developing Alzheimer's disease. can be administered prophylactically to the general population without the need for any assessment of risk to the target patient. The method is particularly useful for individuals with a known genetic risk for Alzheimer's disease. Such individuals include those with relatives who have experienced the disease and those with genetic or biochemical This includes individuals whose risk has been determined by analysis of markers.

[0109] In asymptomatic patients, treatment can begin at any age (e.g., 10, 20, or 30 years of age). However, treatment is usually not given until patients reach the age of 40, 50, 60, or 70. Treatment typically involves multiple doses over a period of time. Treatment may be performed to monitor the response of activated T cells or B cells to the antibody or therapeutic agent over time. If the response decreases, the blood pressure may be increased. The starting dose is indicated.

[0110] In prophylactic applications, pharmaceutical compositions containing tau peptides are administered to treat Alzheimer's disease or other Patients who are susceptible to or otherwise at risk for tauopathy should be screened for the biochemistry of the disease. Clinical, histological and / or behavioral symptoms, its complications and those presented during the development of the disease Eliminate or reduce the risk or severity of diseases, including intermediate pathological phenotypes; and In therapeutic applications, the tau peptide is administered in an amount sufficient to delay the onset of the disease. and administering a pharmaceutical composition containing the compound to a patient suspected of having or already suffering from such a disease. The disease manifestations (biomedicinal, metabolic, and phenotypic) including its complications and intermediate pathological phenotypes in the development of the disease. to cure or at least partially arrest the pathological (chemical, histological, and / or behavioral) Administer in an amount sufficient to stop

[0111] A dose of this invention effective to prevent and / or treat a neurodegenerative disease, disorder, or condition. The pharmaceutical compositions of the invention may be prepared in accordance with the mode of administration, target site, physiological condition of the patient, other drugs administered, and The type of treatment varies depending on many different factors, including the type and whether the treatment is prophylactic or therapeutic. The amount of peptide depends on whether an adjuvant is also administered, and in the absence of an adjuvant Higher doses are required. The timing of injections varies from once daily to once a year to once every 10 years. The typical regimen is immunization, followed by a 6-week Another regimen consists of an immunization followed by booster injections at time intervals such as 100-200 mg / kg. 1, 2, 6, 9, and 12 months later, followed by booster injections. It involves monthly injections. Alternatively, booster injections may be administered depending on the immune response. Can be irregular as indicated.

[0112] Those skilled in the art will appreciate that the regimen for priming and boosting administrations is determined by the measured immune response after administration. It will be readily apparent that the results can be adjusted based on the answer. For example, the boosting composition may be adjusted based on the Generally, weeks or months after administration of the priming composition, e.g., Approximately 2 to 3 weeks, 4 weeks, 8 weeks, 16 weeks, or 2 weeks after administration After 0 weeks, or after 24 weeks, or after 26 weeks, or after 28 weeks, or after 30 weeks , or 32 weeks later, or 36 weeks later, or 1 to 2 years later.

[0113] The peptides may be administered parenterally, topically, intravenously, or orally for prophylactic and / or therapeutic treatment. , by subcutaneous, intraarterial, intracranial, intraperitoneal, intradermal, intranasal, or intramuscular means. The most typical route of administration of immunogenic agents is subcutaneous or intramuscular injection. The latter type of injection is most typically given intramuscularly in the arm or leg.

[0114] According to certain embodiments, one or more booster immunizations may be administered. The antigens in each priming and boosting composition depend on how many boosting compositions are used. Whichever is used, they need not be identical, but may share antigenic determinants or be substantially similar to each other. It should be similar.

[0115] The composition may, if desired, contain one or more unit dosage forms containing the active ingredient. The composition may be presented in a kit, pack, or dispenser. The packing material may, for example, comprise metal or plastic foil, such as a blister pack. The kit, pack, or dispenser may be accompanied by instructions for administration.

[0116] According to a particular embodiment, the kit comprises a pharmaceutical composition comprising a liposome according to an embodiment of the present invention. At least one of the compositions and pharmaceutical compositions comprising a conjugate according to one embodiment of the present invention Also includes one.

[0117] Embodiment The present invention also provides the following non-limiting embodiments.

[0118] Embodiment 1 is a. a tau peptide; b. Helper T cell epitopes and and a tau peptide is displayed on the surface of the liposome.

[0119] Embodiment 2 is the liposome of embodiment 1, wherein the tau peptide is a tau phosphopeptide. It is a room.

[0120] Embodiment 3 is a method for treating a cancer of the liver according to embodiment 1 or 2, further comprising administering toll-like receptor ligands to the cancer. It is a vesicle.

[0121] In embodiment 4, the toll-like receptor ligand is a toll-like receptor 4 ligand and a toll-like receptor 4. The liposome of embodiment 3, comprising at least one of receptor 9 ligands.

[0122] Embodiment 5 is an embodiment in which the toll-like receptor ligand is a toll-like receptor 4 ligand. 3 or 4. The liposome according to claim 3.

[0123] Embodiment 6 is a method for treating a toll-like receptor 4 disease, wherein the toll-like receptor 4 ligand comprises monophosphoryl lipid A (MPLA). , the liposomes described in embodiment 5.

[0124] Embodiment 7 is an embodiment in which the toll-like receptor ligand is a toll-like receptor 9 ligand. 3 or 4. The liposome according to claim 3.

[0125] Embodiment 8 is a method for treating a toll-like receptor 9 disease, wherein the toll-like receptor 9 ligand comprises a lipidated CpG oligonucleotide. , the liposome of embodiment 7.

[0126] Embodiment 9 is a. a tau peptide; b. helper T cell epitopes; ci Toll-like receptor 9 ligand, and ii. Toll-like receptor 4 ligand with at least one of 2. The liposome of embodiment 1, comprising:

[0127] Embodiment 10 is the liposome of embodiment 9, wherein the tau peptide is a tau phosphopeptide. It is Soum.

[0128] Embodiment 11 is a method for treating a toll-like receptor 9 ligand comprising administering to a subject therapies comprising administering to the subject the toll-like receptor 9 ligand is a lipidated CpG oligonucleotide. The liposome of embodiment 9 or 10.

[0129] Embodiment 12 includes a toll-like receptor 4 ligand and a toll-like receptor 9 ligand. 12. The liposome of any one of embodiments 9 to 11.

[0130] Embodiment 13 is directed to a method for treating a toll-like receptor 4 disease, wherein the toll-like receptor 4 ligand comprises monophosphoryl lipid A (MPLA). 13. The liposome of embodiment 12.

[0131] Embodiment 14 is a. tau phosphopeptide; b. helper T cell epitopes; c. lipidated CpG oligonucleotides; d. an adjuvant containing a Toll-like receptor 4 ligand; Including, A liposome in which tau phosphopeptides are displayed on the surface of the liposome.

[0132] Embodiment 15 is directed to a method for treating a toll-like receptor 4 disease, wherein the toll-like receptor 4 ligand comprises monophosphoryl lipid A (MPLA). 15. The liposome of embodiment 14.

[0133] Embodiment 16 is a method for producing a liposome comprising encapsulating a helper T cell epitope in a liposome. , a liposome according to any one of embodiments 1 to 15.

[0134] Embodiment 16a is a liposome in which the helper T cell epitope is incorporated into the membrane of the liposome. , a liposome according to any one of embodiments 1 to 15.

[0135] Embodiment 16b is a method for producing liposomes in which helper T cell epitopes are displayed on the surface of the liposome. , a liposome according to any one of embodiments 1 to 15.

[0136] Embodiment 17 is a. tau phosphopeptide; b. helper T cell epitopes; c. lipidated CpG oligonucleotides; d. Monophosphoryl lipid A (MPLA) and Including, Tau phosphopeptides are displayed on the surface of the liposomes; T cell epitopes are encapsulated within liposomes. A liposome composition.

[0137] Embodiment 17a is a process in which the MPLA is 3-O-deacylated-4'-monophosphoryl lipid A, preferably or MPL™.

[0138] Embodiment 17b is a process for preparing a hydroxyl group-containing lipid comprising: a) a hydroxyl group containing hydroxyl group; b) a hydroxyl group containing hydroxyl group; c) a hydroxyl group containing hydroxyl group; d ... The ribozyme according to embodiment 17, which is preferably 3D-(6-acyl)PHAD®. It is a vesicle.

[0139] Embodiment 17c is a process in which the MPLA is monophosphoryl 3-deacylated lipid A, preferably 3D- The liposome of embodiment 17 is PHAD®.

[0140] Embodiment 18 is directed to 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DM PC), 1,2-dimyristoyl-sn-glycero-3-phosphoryl-3'-rac-glycero one or more selected from the group consisting of glycerol (DMPG), and cholesterol The liposome of any of embodiments 1 to 17c, further comprising a number of lipids.

[0141] Embodiment 19 is a method for treating a tau peptide comprising administering to a subject the method of the present invention ... selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 12. or an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 12. having an amino acid sequence that is at least 85%, 90%, or 95% identical to the sequence, 19. The liposome of any one of Aspects 1 to 18.

[0142] Embodiment 19-1 is a method for treating a tau peptide comprising administering to a subject a tau peptide selected from the group consisting of SEQ ID NOs: 1 to 3 and 5 to 12. 20. The liposome of embodiment 19, which is a phosphopeptide comprising a selected amino acid sequence. is.

[0143] Embodiment 19-2 is an embodiment in which the tau phosphopeptide comprises the amino acid sequence of SEQ ID NO: 1. The liposome is described in Embodiment 19-1.

[0144] Embodiment 19-3 is an embodiment in which the tau phosphopeptide comprises the amino acid sequence of SEQ ID NO:2. The liposome is described in Embodiment 19-1.

[0145] Embodiment 19-4 is an embodiment in which the tau phosphopeptide comprises the amino acid sequence of SEQ ID NO: 3. The liposome is described in Embodiment 19-1.

[0146] Embodiment 19a is a method for preparing a liposome comprising administering to a subject the amino acid sequence of which is a tau peptide displayed on the surface of the liposome. Embodiments 19, 19-1, 19-2, 19-3, 19-4, 19-5, 19-6, 19-7, 19-8, 19-9, 19-10, 19-11, 19-12, 19-13, 19-14, 19-15, 19-16, 19-17, 19-18, 19- 2, 19-3, and 19-4.

[0147] Embodiment 19b is a method in which the one or more modifications are palmitoylation and dodecyl-modified cysteines. The liposome according to embodiment 19a, comprising at least one of:

[0148] Embodiment 19c is a tau peptide modified at its N-terminus by one or more modifications. The liposome of embodiment 19a or 19b, wherein the liposome is

[0149] Embodiment 19d is a tau peptide modified at its C-terminus by one or more modifications. The liposome of any of embodiments 19a to 19c, wherein the liposome is

[0150]

[0023] Embodiments in which the tau peptide is palmitoylated at both its N-terminus and C-terminus. 19e is the liposome of embodiment 19d.

[0151] Embodiment 19f is an embodiment in which the tau peptide comprises one or more modifications to facilitate one or more modifications. 19a to 19e, further comprising one or more additional amino acids. It is a liposome.

[0152] Embodiment 19g is an embodiment in which the one or more additional amino acids are Lys, Cys, Ser, and The liposome of embodiment 19f, wherein the liposome is selected from the group consisting of:

[0153] Embodiment 19h is a method according to embodiment 19, wherein the tau peptide is amidated at its C-terminus. The liposome is described in any one of the above 19g.

[0154] Embodiment 19i is an embodiment wherein the tau peptide is selected from the group consisting of SEQ ID NO:27 to SEQ ID NO:38. 19h. The liposome of any of embodiments 19 to 19h, wherein the liposome consists of an amino acid sequence do.

[0155] Embodiment 19j is a method according to embodiment 1, wherein the tau peptide consists of the amino acid sequence of SEQ ID NO: 27. 9 to 19i.

[0156] Embodiment 19k is a method according to embodiment 1, wherein the tau peptide consists of the amino acid sequence of SEQ ID NO: 28. 9 to 19i.

[0157] Embodiment 19l is a method according to embodiment 1, wherein the tau peptide consists of the amino acid sequence of SEQ ID NO: 29. 9 to 19i.

[0158] In embodiment 20, the helper T cell epitope consists of SEQ ID NO: 23 to SEQ ID NO: 26. 191. The method of claim 1, further comprising administering to said patient at least one amino acid sequence selected from the group consisting of: The liposome is as described above.

[0159] In embodiment 20a, the helper T cell epitopes consist of SEQ ID NO: 23 to SEQ ID NO: 26. 21. The liposome of embodiment 20, comprising at least two amino acid sequences selected from the group consisting of: It is a room.

[0160] In embodiment 20b, the helper T cell epitopes consist of SEQ ID NO: 23 to SEQ ID NO: 26. 21. The liposome of embodiment 20, comprising at least three amino acid sequences selected from the group consisting of: It is a room.

[0161] In embodiment 20c, the helper T cell epitopes are the four sequences of SEQ ID NO: 23 to SEQ ID NO: 26. 21. The liposome of embodiment 20, comprising the amino acid sequence:

[0162] Embodiment 20d is directed to a method comprising administering to a subject a subject a subject having a disease or condition ... Any of embodiments 20a to 20c, wherein the amino acid sequences are covalently linked by a linker. The liposome is described in any one of the above.

[0163] Embodiment 20e is an embodiment in which the linker is selected from the group consisting of Val (V), Ala (A), Arg (R), Gly (G), Ser(S), Lys(K) The liposome of embodiment 20d comprises an acid.

[0164] Embodiment 20f is an embodiment wherein the linker is selected from the group consisting of VVR, GS, RR, and RK. The liposome of embodiment 20e comprises an amino acid sequence as defined in claim 20b.

[0165] Embodiment 20g is a method for producing a human T-helper cell epitope, wherein the T-helper cell epitope is amidated at its C-terminus. A liposome according to any one of embodiments 20 to 20f.

[0166] Embodiment 20h is a method for treating a T-cell tumor comprising administering to a subject the T-cell epitope of which is a T-cell tumor. Depending on the location, the liposomes may be inserted into the membrane, displayed on the surface, or Any of embodiments 20 to 20g, wherein the compound is modified for encapsulation in a hydroxysome. The liposome is described in

[0167] In embodiment 20i, the helper T cell epitopes consist of SEQ ID NO: 13 to SEQ ID NO: 17. The recombinant protein according to any of embodiments 20 to 20h, consisting of an amino acid sequence selected from the group It is a vesicle.

[0168] Embodiment 20j comprises a tau peptide and a helper T cell epitope in a weight ratio of 6:1. 20i。 20i. The liposome of any one of embodiments 1 to 20i.

[0169] Embodiment 20k comprises a tau peptide and a helper T cell epitope in a weight ratio of 5:1. 20i。 20i. The liposome of any one of embodiments 1 to 20i.

[0170] Embodiment 201 comprises a tau peptide and a helper T cell epitope in a weight ratio of 4:1. 20i。 20i. The liposome of any one of embodiments 1 to 20i.

[0171] Embodiment 20m comprises a tau peptide and a helper T cell epitope in a weight ratio of 3:1. 20i。 20i. The liposome of any one of embodiments 1 to 20i.

[0172] Embodiment 20n comprises a tau peptide and a helper T cell epitope in a 2:1 weight ratio. 20i。 20i. The liposome of any one of embodiments 1 to 20i.

[0173] Embodiment 20o is a composition comprising a tau peptide and a helper T cell epitope in a 1:1 weight ratio. 20i。 20i. The liposome of any one of embodiments 1 to 20i.

[0174] In embodiment 21, the lipidated CpG oligonucleotide is selected from SEQ ID NO: 18 to SEQ ID NO: 22. 20. The method of any one of embodiments 1 to 20, comprising a nucleotide sequence selected from the group consisting of: The liposomes are as described above.

[0175] Embodiment 21a is a method for preparing a CpG oligonucleotide comprising one or more phosphorothioates. 22. The liposome of embodiment 21, having internucleotide linkages.

[0176] Embodiment 21b is a method for preparing a CpG oligonucleotide comprising all phosphorothioate nucleotides. The liposome of embodiment 21a has inter-column bonds.

[0177] Embodiment 21c is an embodiment in which the lipidated CpG oligonucleotide is at least 21. The method of claim 20, further comprising covalently linking a CpG oligonucleotide to a lipophilic group. 21b.

[0178] Embodiment 21d is an embodiment in which the linker comprises (C2H4O)n, where n is an integer from 0 to 10.

[0042] The liposomes of embodiment 21c.

[0179] Embodiment 21e is an embodiment wherein the linker comprises an alkyl spacer having 3 to 12 carbons. The liposomes of embodiment 21c.

[0180] Embodiment 21f is an embodiment of embodiment 21, in which at least one lipophilic group is cholesterol. 21e.

[0181] Embodiment 21g is an embodiment in which the lipidated CpG oligonucleotide is (C2H4O)n, wherein: and n is an integer of 3 to 5. 21f. The nucleotide sequence of SEQ ID NO: 18 or SEQ ID NO: 19. The liposome according to any one of the above items.

[0182] Embodiment 22 is a. Tau having an amino acid sequence selected from the group consisting of SEQ ID NO: 27 to SEQ ID NO: 38 A peptide, b. A herbicidal drug having an amino acid sequence selected from the group consisting of SEQ ID NO: 39 to SEQ ID NO: 44 per T cell epitope, preferably selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO: 17 a helper T cell epitope consisting of an amino acid sequence c. having a nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 22; containing one or more phosphorothioate internucleotide linkages and a lipidated CpG oligonucleotide covalently linked to at least one cholesterol; d. Monophosphoryl lipid A (MPLA) and The liposome comprises:

[0183] Embodiment 22a is a. A tau gene comprising the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29 Suphopeptides and b. a helper T cell epitope consisting of the amino acid sequence of SEQ ID NO: 13; c. A linker containing (C2H4O)n [wherein n is an integer of 3 to 7] is used to connect the from the nucleotide sequence of SEQ ID NO: 18 or SEQ ID NO: 19 covalently bound to a sterol a lipidated CpG oligonucleotide comprising d. Monophosphoryl lipid A (MPLA) and 23. The liposome of embodiment 22, comprising:

[0184] Embodiment 22b is an embodiment in which the MPLA is 3-O-deacylated-4'-monophosphoryl lipid A, preferably or MPL™.

[0185] Embodiment 22c is an embodiment in which the MPLA is preferably 3D-(6-acyl)PHAD® The liposome of embodiment 22 or 22a, wherein

[0186] Embodiment 22d is an embodiment in which the MPLA is preferably 3D-PHAD®. The liposome according to embodiment 22 or 22a.

[0187] Embodiment 23 is a method for producing a liposome comprising encapsulating a helper T cell epitope in a liposome. , a liposome according to any one of embodiments 22 to 22d.

[0188] Embodiment 24 is a method for preparing a liposome according to any one of embodiments 1 to 23, comprising administering to a subject a pharmaceutically acceptable carrier. and a carrier.

[0189] Embodiment 25 relates to a compound having the following structure:

[0190] [ka] [In the formula, x is an integer from 0 to 10, n is an integer from 2 to 15. and an immunogen conjugated thereto via a linker, and a hydroxyl group-containing carrier.

[0191] Embodiment 25a relates to a compound having the structure of formula (II):

[0192] [ka] [In the formula, x is an integer from 0 to 10, n is an integer from 2 to 15. and an immunogen conjugated thereto via a linker, and a hydroxyl group-containing carrier.

[0193] Embodiment 26 is the method of any of embodiments 25 or 25a, wherein x is an integer from 2 to 6. The conjugate is as described above.

[0194] Embodiment 27 is a conjugate of any of embodiments 25 or 25a, wherein x is 3. It is a gate.

[0195] Embodiment 28 is a compound according to any one of embodiments 25 to 25a, wherein n is 3 to 7. It's Jugate.

[0196] Embodiment 29 is a method for treating a pulmonary edema, wherein the carrier is keyhole limpet hemocyanin (KLH), tetanus toki Pseudomonas aeruginosa, CRM197, and a mixture of outer membrane proteins from N. meningitidis an immunogenic carrier selected from the group consisting of an immunoglobulin M (OMP), an immunoglobulin M (OMP), or a derivative thereof; 29. The conjugate according to any one of embodiments 25 to 28.

[0197] Embodiment 30 is a method for preparing a tau phosphopeptide comprising administering to a subject the tau phosphopeptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 12. 30. The conjugate according to any of embodiments 25 to 29, consisting of an amino acid sequence selected from It is.

[0198] Embodiment 30a is the method wherein the tau phosphopeptide is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 30a. 31. The conjugate of embodiment 30, consisting of the amino acid sequence of:

[0199] Embodiment 31 is any of embodiments 25 to 30, wherein the carrier is CRM197. It is a conjugate of

[0200] Embodiment 32 is directed to a compound having the following structure:

[0201] [ka] [wherein n is 3 to 7] 26. The conjugate of embodiment 25, having the formula:

[0202] Embodiment 32a is KLH-[m-maleimidobenzoyl-N-hydroxysuccinimide ester-cis tein-(C2H4O)x-tau peptide] n

[0203] [ka] [In the formula, the tau peptide consists of SEQ ID NO: 1 or SEQ ID NO: 3; x is an integer from 0 to 10, n is an integer from 2 to 15. 26. The conjugate of embodiment 25, wherein

[0204] Embodiment 33 relates to a conjugate according to any one of embodiments 25 to 32a and a pharmaceutical and a carrier acceptable thereto.

[0205] Embodiment 33a is a pharmaceutical composition according to claim 33, further comprising an adjuvant. .

[0206] Embodiment 33b is an embodiment in which the adjuvant is a sachet of a TLR-4 ligand and a TLR-9 ligand. The pharmaceutical composition according to claim 33a comprises at least one of the following:

[0207] Embodiment 34 provides administering to a subject at least one of the pharmaceutical compositions of embodiments 24 and 33 to 33b. and administering at least one of the following to a subject suffering from a neurodegenerative disorder: How to induce.

[0208] Embodiment 35 relates to administering to a subject the vaccine of any one of embodiments 24 and 33 for priming immunization. 3b. Administering at least one of the pharmaceutical compositions of 3a. to a subject and administering a booster immunization to the subject. administering at least one of the pharmaceutical compositions of embodiments 24 and 33 to 33b for 35. The method of embodiment 34, comprising administering.

[0209] Embodiment 36 provides a method for administering to a subject at least one of the pharmaceutical compositions of embodiment 24 or 33. and administering to a subject in need thereof one or more of the following: It is a method of treatment or prevention.

[0210] Embodiment 37 relates to administering to a subject the vaccine of any one of embodiments 24 and 33 for priming immunization. 3b. Administering at least one of the pharmaceutical compositions of 3a. to a subject and administering a booster immunization to the subject. administering at least one of the pharmaceutical compositions of embodiments 24 and 33 to 33b for 37. The method of embodiment 36, comprising administering.

[0211] Embodiment 38 is a method for treating a neurodegenerative disease or disorder caused by the formation of neurofibrillary lesions. 38. The method according to any of embodiments 34 to 37, wherein the method is carried out or associated with do.

[0212] Embodiment 39 is a method for treating a neurodegenerative disease or disorder, including Alzheimer's disease, Parkinson's disease, Chlamydia trachomatis, or Chlamydia trachomatis. Leutzfeldt-Jakob disease, dementia pugilistica, Down syndrome, Gerstmann-Straussler - Scheinker's disease, inclusion body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury Harm, amyotrophic lateral sclerosis, Parkinson-dementia complex of Guam, with neurofibrillary tangles Non-Guam motor neuron disease, argyrophilic grain dementia, corticobasal degeneration, Lewy cognition amyotrophic lateral sclerosis, diffuse neurofibrillary tangles with calcification, frontotemporal cognition dementia, preferably frontotemporal dementia with parkinsonism linked to chromosome 17 (FT DP-17), frontotemporal dementia, Hallevorden-Spatz disease, multi- System atrophy, Niemann-Pick disease type C, Pick's disease, progressive subcortical gliosis, progressive nuclear Epigastric paresis, subacute sclerosing panencephalitis, dementia tangles only, postencephalitic parkinsonism, muscle tension dystrophy, chronic traumatic encephalopathy (CTE), primary age-related tauopathy (PAR 39. The method of any of embodiments 34 to 38, wherein the patient is a patient with dementia with Lewy bodies (LBD), or a patient with dementia with Lewy bodies (LBD). This is the method described above.

[0213] Embodiment 40 is a method for treating a neurodegenerative disease or disorder, including Alzheimer's disease, Parkinson's disease, or Diabetes Mellitus. Ung's syndrome, progressive supranuclear palsy (PSP), and frontotemporal dementia are linked to chromosome 17 Parkinsonism (FTDP-17), Pick's disease, corticobasal degeneration, Lewy dementia , amyotrophic lateral sclerosis, myotonic dystrophy (dysphasia), chronic traumatic encephalopathy (CT E), cerebrovascular disease, primary age-related tauopathy (PART), or dementia with Lewy bodies ( 40. The method of any of embodiments 34 to 39, wherein the IL-16 receptor agonist (IL-16 receptor agonist) is a IL-16 receptor agonist (IL-16 receptor agonist).

[0214] Embodiment 40b is a method for treating a neurodegenerative disease or disorder, comprising administering to a subject a therapeutically effective amount of ... (PSP), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP- 17), or Pick's disease, and PART (primary age-related tauopathy), 40. The method of any one of aspects 34 to 39.

[0215] Embodiment 40c is an embodiment in which the neurodegenerative disease or disorder is Alzheimer's disease, Parkinson's disease, Down syndrome, frontotemporal dementia and chromosome 17-linked parkinsonism (FTDP) -17), corticobasal degeneration, Lewy dementia, amyotrophic lateral sclerosis, myotonic dystonia Dysphagia, chronic traumatic encephalopathy (CTE), cerebrovascular disease, primary age-related tau 39. The patient is a patient with autism spectrum disorder (PART), or a Lewy body dementia (LBD). The method is any one of the above.

[0216] Embodiment 41 is a pharmaceutical composition of embodiment 24 and embodiment 33, 33a, or 33b. b) a kit comprising at least one of the pharmaceutical compositions of b).

[0217] Embodiment 42 is an amino acid sequence selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO: 17. It is a helper T cell epitope consisting of

[0218] Embodiment 43 is a pharmaceutical composition comprising a helper T cell epitope according to embodiment 42. be.

[0219] Embodiment 44 comprises administering to a subject an antigen together with the pharmaceutical composition of embodiment 43. and a method for enhancing an immune response to an antigen in a subject in need thereof, comprising: . [Example]

[0220] The following examples of the present invention are intended to further illustrate the nature of the present invention. The examples do not limit the invention, the scope of which is determined by the appended claims. I would like you to understand this.

[0221] All experimental methods used in the following examples are conventional unless otherwise specified. All reagents used in the following embodiments are purchased from standard reagent suppliers unless otherwise specified. It was purchased from.

[0222] [Example 1] Preparation of liposomal vaccines Preparation of control liposomal vaccine (ethanol injection technique) The control liposomal vaccine was administered by ethanol (EtOH) injection technique followed by extrusion. First, DMPC (Lipoid GmbH, Ludwigshafen, Germany) was used. fen), DMPG (Lipoid GmbH, Ludwigshafen, Germany), Lactosterol (Dishman, Netherlands), and MPLA (Avanti Pola Lipids, Alabama, USA) in a molar ratio of 9:1:7:0.05 with EtOH and and tert-butanol (t-BuOH) in a 20:1 (V / V) mixture at 60°C. The lipid / ethanol solution was dissolved in phosphate buffer to maintain a 10% EtOH concentration. Diluted in physiological saline (PBS), pH 7.4, at 60°C, and multilamellar liposome vesicles (MLVs) Subsequently, EmulsiFlex-C5 (Avestin, Canada) was used. ) to insert the MLVs into three polycarbonate filters in series with 0.08 μm pore size. The resulting liposomes were subjected to five successive extrusions through a Whatman filter. The mixture was diluted with PBS, pH 7.4, and heated to 60°C before adding the tau peptide. A liposome solution was obtained.

[0223] Tetrapalmitoyl acetate of SEQ ID NO: 2, hereinafter referred to as the active pharmaceutical ingredient (API), Acetate tetrapalmitoylated phosphorylated tau peptide (Bachem AG, Sumitomo Dainippon Pharma, Tokyo, Japan) The solution was diluted with 2.0% octyl β-D-glucopyranoside (Sigma) at a concentration of 1 mg / mL. The peptide solution was dissolved in PBS (pH 11.4) containing α-Aldrich (USA). The liposome solution was then injected at 60°C and stirred for 30 minutes at 60°C. The final volume was obtained by ultrafiltration, and buffer exchange was performed with PB during diafiltration. The procedure was repeated 10 times using 100% ethanol at pH 7.4. After that, the API was presented on the surface of the liposomes. The resulting liposomes were filtered through two 0.2 μm polycarbonate syringe filters in series. The solution was sterile filtered through a filter and the final product was stored at 5°C.

[0224] Liposomes X, Y, Z, and Z + Vaccine preparation Liposomal X and Y vaccines are prepared using a lipid thin layer technique followed by homogenization and It was produced by extrusion molding.

[0225] It has a final API concentration of 1200ug / ml and a final T50 concentration of 1200ug / ml. Liposome Z + The vaccine is produced by an ethanol injection technique followed by extrusion. , with a final API concentration of 400ug / ml and a final T50 concentration of 100ug / ml Liposomal Z vaccine is produced using a lipid thin layer technique followed by homogenization and extrusion. Generated by.

[0226] With a final API concentration of 400ug / ml and a final T50 concentration of 400ug / ml Liposome Z ++ The vaccine is prepared using a lipid thin layer technique followed by homogenization and extrusion. Produced by molding.

[0227] With a final API concentration of 1200ug / ml and a final T50 concentration of 300ug / ml Liposome Z +++ The vaccine is produced by an ethanol injection technique followed by extrusion. did.

[0228] Liposomes X, Y, Z, and Z by lipid thin layer technique ++ Vaccine preparation Liposomes X, Y, Z, and Z ++ The vaccine is prepared using a lipid thin layer technique followed by homogenization. First, DMPC (Lipoid GmbH) was used for the preparation of the DMPC polymer. H, Ludwigshafen, Germany), DMPG (Lipoid GmbH, Germany) udwigshafen), cholesterol (Dishman, The Netherlands), and mono Phosphoryl hexa-acyl lipid A3-deacylation synthesis (3D-(6-acyl)PHAD) (Avanti Polar Lipids, Alabama, USA) in a 9:1: The liposomes were solubilized in EtOH at a molar ratio of 7:0.05 at 60°C, except for liposome Y, which was dissolved in 3D -(6-acyl)PHAD® was not contained. Ethanol was removed under vacuum. Evaporation in an otavapor resulted in a thin lipid layer.

[0229] The lipid film was coated with 0.15 mg / mL of T50 peptide (Peptides & Epilep PBS, pH 7.4, 5% DMSO (all Sigma-Aldrich, Germany) The sample was rehydrated using a lipid thin film (Aldrich). The sample was gently agitated for 15 minutes to form a thin lipid layer. The resulting multilamellar vesicles were subjected to 10 freeze-thaw cycles. The cells were then subjected to a cycle (liquid N2 and a water bath at 37°C) for homogenization and then diluted to 0.0 Continuous extrusion through a polycarbonate membrane with 8 um pore size (Whatman, UK) Both the homogenization and extrusion steps were carried out by Emulsi. The assay was performed using a Flex-C5 (Avestin, Canada). The extruded liposomes containing the tide were concentrated by ultrafiltration and then filtered through a diafiltration tray. The buffer was exchanged into PBS, pH 7.4 by filtration. The resulting liposomes were washed with PBS, pH 7.4. Dilute with H7.4 and heat to 60°C before adding tau peptide and adjuvant. A liposome solution of 1000 mg was obtained.

[0230] CpG2006-Cholesterol (CpG2006-Chol) (Microsynt h, Switzerland) has all internucleotide bonds as thiophosphates, and The end is modified with a cholesterol molecule via a phosphate bond via a PEG spacer. CpG2006-cholesterol (CpG2 006-Chol) (Microsynth, Switzerland) in PBS, pH 7.4 at 1 mg / Dissolve in mL and inject into liposome solution (liposome X is an exception, CpG2006-C hol), and then after 15 minutes of incubation, the API Inserted.

[0231] API (Bachem AG, Switzerland) was added to 2% octyl β- PBS, pH 11, containing D-glucopyranoside (Sigma-Aldrich, USA). The peptide solution was dissolved in 4 and injected into the liposome solution at 60°C. The mixture was stirred at 0°C. Concentration was performed by ultrafiltration to a target value (40 for liposomes X, Y, and Z). 0ug / ml API and 100ug / ml T50, Liposomal Z ++ So 400u 100µg / ml API and 400µg / ml T50) and buffer exchange was performed The filtration was performed 10 times with PBS, pH 7.4. The resulting liposomes displayed on the surface of the membrane were then placed on a 0.2 μm polycarbonate syringe. The solution was sterile filtered through a di-filter and the final product was stored at 5°C.

[0232] Preparation of liposomes by ethanol injection technique The liposomal O vaccine was prepared by ethanol (EtOH) injection technique followed by extrusion. First, DMPC (Lipoid GmbH, Ludwigshafen, Germany) was used. en), DMPG (Lipoid GmbH, Ludwigshafen, Germany), Sterol (Dishman, Netherlands), and MPLA (Avanti Polar Lipids, Alabama, USA) in a molar ratio of 9:1:7:0.05 with EtOH and Soluble in a 20:1 (V / V) mixture of tert-butanol (t-BuOH) at 60°C The lipid / ethanol solution was diluted with phosphate buffer to maintain a 10% EtOH concentration. Diluted in physiological saline (PBS), pH 7.4, at 60°C, to form multilamellar liposome vesicles (MLVs). Then, EmulsiFlex-C5 (Avestin, Canada) Using MLVs, fill three polycarbonate sheets in series with 0.08 μm pore size. The resulting liposomes were subjected to five successive extrusions through a Whatman extruder. The mixture was diluted with PBS, pH 7.4, and heated to 60°C to prepare the lysate before adding the tau peptide. A liposome solution was obtained.

[0233] T46 peptide (Pepscan, Netherlands) at 1 mg / mL in PBS, pH 7.4 After dissolving and injecting into the liposome solution, and then incubating for 15 minutes, AP I inserted.

[0234] API (Bachem, Switzerland) was added to 2% octyl β-D-glucan at a concentration of 1 mg / mL. Dissolved in PBS, pH 11.4, containing guanidine diphosphate (Sigma-Aldrich, USA). However, the peptide solution was injected into the liposome solution at 60°C, and then the mixture was heated at 60°C for 30 minutes. Concentration was performed by ultrafiltration to target values ​​(400 μg / ml API and 10 0 μg / ml T46), and buffer exchange was performed with PBS during diafiltration, The procedure was repeated 10 times using pH 7.4. The API was then presented on the surface of the liposomes. The resulting liposomes were filtered through a 0.2 μm polycarbonate syringe filter. The mixture was sterile filtered and the final product was stored at 5°C.

[0235] Liposome Z by ethanol injection + and Liposome Z +++ Vaccine preparation Liposome Z + and Liposome Z +++ The vaccine is based on an ethanol injection process. First, DMPC (Lipoid GmbH, Ludwigshafen, Germany) was used. DMPG (Lipoid GmbH, Ludwigshafen, Germany) n), cholesterol (Dishman, Netherlands), and 3D-(6-acyl)PH AD® (Avanti Polar Lipids, Alabama, USA) was used in an amount of approximately The T50 peptide (Ba chem AG, Switzerland) in 10 mM His / 270 mM sucrose (pH 5.8 The lipid ethanol solution was then dissolved in a solution containing T50 peptide. The resulting solution was injected into the oocyte and gently stirred for 15 minutes to generate multilamellar vesicles (MLVs). genization (liposome Z + So, 6 times, Liposome Z +++ So homogenization without any addition), followed by a polycarbonate membrane with a pore size of 0.08 μm (Whatman, Continuous extrusion molding (Liposome Z) through + 5 times, Liposome Z +++ So 3~ 5 times). Liposome Z + Now, the homogenization and extrusion steps Both procedures were performed using an EmulsiFlex-C5 (Avestin, Canada). Room Z +++ The extrusion was carried out using a LIPEX filter extruder. The formed liposomes were concentrated by ultrafiltration and diluted by diafiltration. The buffer was exchanged to 20 mM His / 145 mM NaCl, pH 7.4. The resulting liposomes with the modified T50 peptide were incubated in 20 mM His / 145 mM NaCl. Dilute with CL, pH 7.4, heat to 60°C, add API and adjuvants The former liposome solution was obtained.

[0236] CpG2006-Chol (Liposome Z + Microsynth, Switzerland, Lipo Some Z +++ (Avecia, USA) at 1 mg / mL of 20 mM His / 145mM Dissolve in 100 mM NaCl, pH 7.4, inject into the liposome solution, and then incubate for 15 minutes. After the incubator, the API was inserted.

[0237] API (Bachem AG, Switzerland) was diluted with 1% octyl β- Carbonate buffer, pH 1, with D-glucopyranoside (Sigma-Aldrich, USA) The peptide solution was dissolved in 0.2% ethanol and heated at 60°C to form liposome Z. + Inject into the solution and then The peptide solution was stirred at 60°C for 1 minute using T-Line Mixing. Liposome Z + The solution was mixed and then stirred for 30 minutes at 60°C. The target value (liposome Z) was obtained by filtering + 1200ug / ml API and 120 0ug / ml T50, Liposomal Z +++ 1200ug / ml API and 30 0 μg / ml) and buffer exchange was performed with 10 mM 10 times with 270 mM His / 270 mM sucrose, pH 6.5. I was displayed on the surface of the liposome. + Liposomes and API are The resulting Z presented on the surface +++ Liposomes were placed in a 0.2 μm polycarbonate syringe. The final product was stored at 5°C after sterile filtration through a capsule filter.

[0238] Preparation of liposomal L, M, and N vaccines Liposomal L, M, and N vaccines are prepared using the lipid thin layer technique followed by homogenization. First, DMPC (Lipoid GmbH, Germany) was prepared by extrusion molding. Ludwigshafen, Germany), DMPG (Lipoid GmbH, Ludwigshafen, Germany) gshafen), cholesterol (Dishman, The Netherlands), and MPLA (A vanti Polar Lipids, Alabama, USA) in a ratio of 9:1:7:0.05 The ethanol was evaporated in a rotavapor under vacuum. By evaporating the solution, a thin lipid layer was obtained.

[0239] Lipid films were prepared in PBS, pH 7.4, 5% DMS containing either O (all Sigma-Aldrich): Liposome M contains 0.15 mg / mL of T48 peptide (Peptides & Elep hants, Germany), or Liposome L 0.13 mg / mL T50 peptide (Peptides & Elephas nts, ​​Germany), or Liposome N contains 0.15 mg / mL of T52 peptide (Peptides & Elep hants, Germany).

[0240] The sample was gently agitated for 15 minutes and then vortexed vigorously to dissolve the lipid film. The resulting multilamellar vesicles were subjected to 10 freeze-thaw cycles (liquid N2 and water bath at 37°C). The mixture was subjected to homogenization and then passed through a polycarbonate membrane ( The mixture was subjected to continuous extrusion through a Whatman (UK). Both extrusion steps were performed using EmulsiFlex-C5 (Avestin, Canada). The extruded liposomes were concentrated by ultrafiltration and then placed in a diafiltration tray. The buffer was exchanged to PBS, pH 7.4 by filtration. The resulting liposomes carrying the T50 or T52 peptide were diluted with PBS, pH 7.4. The mixture was heated to 60°C to obtain a liposome solution before adding the tau peptide.

[0241] API (Bachem AG, Switzerland) was added to 2% octyl β- PBS, pH 11, containing D-glucopyranoside (Sigma-Aldrich, USA). The peptide solution was dissolved in 4 and injected into the liposome solution at 60°C. The mixture was stirred at 0°C. Concentration was performed by ultrafiltration to the target values ​​(API of 400 μg / ml and and 100µg / ml T48, T50, or T52), and buffer exchange was performed using PBS, pH 7.4 was used during filtration 10 times. The resulting liposomes displayed on the surface of the liposome were placed on a 0.2 μm polycarbonate silicone gel. The solution was sterile filtered through a syringe filter and the final product was stored at 5°C.

[0242] Preparation of liposomal R, S, and T vaccines Liposomal R, S, and T vaccines are delivered via an ethanol injection-based process followed by extrusion. First, DMPC (Lipoid GmbH, Ludwigshafen, Germany) was used for the production of DMPC. dwigshafen), DMPG (Lipoid GmbH, Ludwigshafen, Germany) afen), cholesterol (Dishman, The Netherlands), and 3D-(6-acyl ) PHAD® (Avanti Polar Lipids, Alabama, USA) The liposomes were solubilized in EtOH at a molar ratio of 9:1:7:0.04 at 60°C. At T, the lipid ethanol solution was diluted to 270°C to reach 10% solvent (EtOH). Mix with 10 mM histidine, pH 5.8, supplemented with 30 mM sucrose, and then The mixture was incubated at 60°C for 1 minute. Liposome S contained T50 peptide (Bachem AG, Switzerland) in 10 mM His / 270 mM sucrose (pH 5.8-6.0) The lipid-buffer mixture for liposomes R, S, and T was gently dissolved in 100 ml of PBS for 15 minutes. The resulting multilamellar vesicles (MLVs) were collected by EmulsiFle x-C5 high-pressure system (Avestin, Canada) with a pore size of 0.08 μm It was subjected to extrusion molding (5 times) through polycarbonate membranes (Whatman, UK).

[0243] The extruded liposomes were concentrated by ultrafiltration and purified by diafiltration. The buffer was then exchanged to 20 mM His / 145 mM NaCl, pH 7.4. The resulting liposomes with encapsulated T50 (liposome S), as well as the resulting liposomes Rooms R and T were further diluted with 20 mM His / 145 mM NaCL, pH 7.4. and heated to 60°C to obtain the liposomal solution before adding the API and T57 (liposomal T). A soluble solution was obtained.

[0244] For liposomal T, T57 was prepared by dissolving 1% octyl β-D-glucopyranoside in deionized distilled water. The solution was dissolved in 1 mg / mL of PEG-400 (Sigma-Aldrich, USA) and inserted into liposomes. After incubation at 60°C for 15 minutes, API insertion was performed.

[0245] API (Bachem AG, Switzerland) was diluted with 1% octyl β- Carbonate buffer, pH 1, with D-glucopyranoside (Sigma-Aldrich, USA) The peptide solution was dissolved in 0.2% ethanol and mixed into the liposome solution at 60°C for 30 minutes. and stirring at 60° C. Concentration was performed by ultrafiltration to obtain the following target values: -1200ug / ml API for Liposome R, -Liposome S has 1200ug / ml API and 300ug / ml T50, Ravini -Liposomal T is 1200ug / ml API and 300ug / ml T57.

[0246] Buffer exchange was performed with 10 mM His / 270 mM sucrose during diafiltration. The procedure was repeated 10 times using a pH 6.5 solution. After that, the API was presented on the surface of the liposomes. The resulting liposomes were filtered through a 0.2 μm polycarbonate syringe filter. The mixture was sterile filtered and the final product was stored at 5°C.

[0247] [Example 2] Preparation of conjugate vaccines Peptides and adjuvants Two multiply phosphopeptide epitopes (three and two phosphorylated peptides, respectively) The sequences of TAUVAC-p7.1 and TAUVAC-p22.1 (containing amino acids) were It is also designed to bind better to cell surface immunoglobulins and to interact with human HLA class I The sequence must contain epitopes predicted to bind with high affinity to A, B, and C molecules. The latter criterion was refined by optimizing the length of the graft to prevent significant nerve damage. CD8 cytotoxicity against tau, which may lead to + To avoid the induction of T cell responses Immune Epitope Database and Analysis Resources Tope Database and Analysis Resources Using a cytoplasmic epitope prediction tool, peptide TAUVAC-p7.1 was found to bind to human HLA clusters. Binds with high affinity to HLA class IA, B, C and HLA class II DQ and DR molecules While the peptide TAUVAC- p22.1 binds to HLA class II DQ and DR molecules with moderate / high affinity It was predicted to contain the epitope (data not shown).

[0248] The phosphorylated tau peptides used in this study (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3) was produced synthetically (Pepscan, The Netherlands) and a phospho residue was added during synthesis. The amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 covalently linked to the KLH carrier via a linker was Conjugates comprising phosphorylated tau peptides having the following sequences are referred to herein as These are called conjugates B and C. These are conjugates that are linked to the CRM carrier via a linker. a conjugate comprising a phosphorylated tau peptide having the amino acid sequence of SEQ ID NO: 2 covalently linked thereto; The gate is referred to herein as Conjugate A.

[0249] To prepare conjugates B and C, the vaccine peptide was lysed in a m-maleimide-based Benzoyl-N-hydroxysuccinimide ester (MBS) linker and peptide It was conjugated to the carrier protein KLH via an extra cysteine ​​at the N-terminus. After removing unbound peptide using an ephadex G25 column, the conjugate After mixing the conjugates, a potent multicomponent adjuvant (Sig ma Adjuvant System, Sigma-Aldrich) or single components Element depot adjuvant (aluminum hydroxide, Alhydrogel®, I The mice were injected into either a vein or a tube (nvivogen) according to the manufacturer's instructions.

[0250] The vaccine peptide was then injected into a vaccinated animal using polyethylene glycol (PEG)-cysteine-acetamide (ACE) Conjugated to the carrier protein CRM197 via a propionamide linker The phosphorylated tau peptide having the amino acid sequence of SEQ ID NO: 2 was synthesized (P Polypeptide Laboratories SAS), phospho residues and PEG A spacer was added during synthesis. Conjugate A contained the carrier protein CRM197. , succinimidyl 3-(bromoacetamido)propionate (SBAP) linker The peptide was prepared by conjugation to the N-terminal cysteine ​​of the peptide via . SBAP was added to CRM197 protein primary amines (-N) via NHS ester reaction chemistry H2). Excess SBAP linker was removed by ultrafiltration and diafiltration. The CRM197-SBAP intermediate was removed using UF / DF. After the reaction was complete, an excess of L-cystine was added. The conjugation reaction was stopped by adding HCl to quench the reaction. The peptide conjugate product was then purified using a Capto Q ImpRes (GE Healthcare Purified using a (care) chromatography column and eluted using an isocratic salt method The purified CRM197-peptide product was then purified using UF / DF. to a concentration of 0.5 mg / mL in 0 mM Tris, 250 mM sucrose, pH 8.1 10% PS80 stock buffer was added to give a final concentration of 0.01% PS80. The CRM197-tau peptide drug substance (DS) was produced by Mix thoroughly before filtering.

[0251] [Example 3] The vaccine induced tau phosphopeptide-specific IgG antibodies All animal experiments were approved and conducted in accordance with local regulations regarding animal testing. Rhesus macaques (Macaca mulatta) are housed at Kunming Biomed International tional Ltd, China, Yunnan Yinmore Bio-Tech Co LTD, China, and Yunnan Laboratory Primates I The animals were obtained from NC, China. The animals were 2-5 years old at the start of immunization and had a minimum weight of 2.5 kg. A detailed clinical examination was performed before the start of treatment and weekly thereafter. Additionally, the macaques were observed twice daily and clinical signs were recorded.

[0252] Adult rhesus macaques (n = 3 males and 3 females / group) were incubated with 1800 μg of sequence Control liposome vaccine with acetate tetrapalmitoylated phosphorylated tau peptide / dose of No. 2 tin (having tetrapalmitoylated phosphorylated tau peptide of SEQ ID NO: 2 and MPLA liposomes) or liposomal vaccines according to embodiments of the present application, e.g., liposomes MuZ (tetrapalmitoylated phosphorylated tau peptide of SEQ ID NO: 2, 3D-(6-acyl) PHAD®, lipidated CpG oligonucleotide CpG2006, and T-cell Liposomes with cytosine peptide T50), or 15 μg / dose of one embodiment of the present invention Conjugate vaccines (e.g., conjugate A, linked to CRM197) Phosphorylated tau peptide (SEQ ID NO: 2) was used on days 1, 29, and 85. The mice were immunized subcutaneously with the CpG oligonucleotide CpG2006 and co-injected with the IgG1 gene. Bleeding was performed before immunization and at 8, 22, 36, 50, 64, 78, 92, 106, and 12 Tests were performed on days 0, 134, and 148 and serum was isolated.

[0253] The specific IgG antibody titer was measured using the phosphorylated tau peptide of SEQ ID NO: 2 as a coating antigen. Sera from individual immunized monkeys were assayed using ELISA. Serial dilutions were made in buffer (PBS, 0.05% Tween 20, 1% BSA) to identify the relevant peptides. The plate was then applied to a 96-well plate coated with PEG-400. After 2 hours of incubation, Afterwards, the sample was removed and the plate was washed with PBST (PBS, 0.05% Tween 20). The antibodies were purified using anti-monkey IgG (KPL) conjugated with HRP, followed by ABT. Detection was performed using S substrate (Roche). All samples were run in eight two-fold dilutions. Each plate included positive and negative control samples. Data were collected per group. Point titers (last serum dilution inducing a positive response) were expressed as the geometric mean.

[0254] As shown in Figure 4, both liposomal Z vaccine and conjugate A were significantly higher than the control liposomal Z vaccine. The phosphopeptide-specific IgG titers were higher than those of the phosphosome vaccine.

[0255] [Example 4] The vaccine induced antibodies specific to pathological tau structures in the human brain All brain tissue was collected from the Netherlands Brain Bank (NBB) for the use of brain autopsies and their clinical information for research purposes. The samples were collected from donors after signing an informed consent for the study. Health), Alzheimer's disease (AD), frontotemporal dementia with tau pathology (FTD-tau), Pick's disease, primary age-related tauopathy (PART), and progressive supranuclear palsy (PSP) Paraffin sections from the parietal cortex, middle frontal gyrus, hippocampus, or The caudate nucleus was included.

[0256] Specifically, control human subjects (healthy) and human subjects with Alzheimer's disease (A Formalin-fixed, paraffin-embedded sections from the parietal cortex of D Braak V / VI were Post-immunization macaque blood diluted 1:100 in conventional immunologic diluent The sections were then washed and stained with goat anti-monkey HRP (Abcam). Finally, a brown specific stain was precipitated in the presence of horseradish peroxidase (HRP). Visualize staining using 3,3'-diaminobenzidine (DAB, Dako) The slides were counterstained with hematoxylin, dehydrated, and mounted in Quick D mounting medium (Kli The images were taken using a Leica DC500 microscope. Ta.

[0257] The results in Figure 5 show that Liposome Z (tetrapalmitoylated phosphorylated tau peptide of SEQ ID NO: 2) 3D-(6-acyl) PHAD®, lipidated CpG oligonucleotide C pG2006, and liposomes carrying the T cell peptide T50) Post-immune serum from rhesus macaques stained pathological tau structures in human brain sections. Serum from rhesus macaques on day 106 after primary immunization with improved liposomes. The macaques had been immunized 0, 1, and 3 months prior to serum collection. Left (AD Braak V / VI) panel shows staining of parietal cortex from a Braak stage V donor. Colors indicate tau tangle staining. Arrows indicate tau tangle staining. Right (healthy) panel shows Braak stage 0 Staining of parietal cortex from a donor is shown. Serum was applied to the sections at a dilution of 1:100, and then Goat anti-monkey antibody was applied at 1:100 in the situ hybridization assay, and staining was visualized using DAB chromogen.

[0258] The results in Figure 6 show that the phosphorylated tau peptide of SEQ ID NO: 2 + soluble CpG and alum water Serum from rhesus macaques immunized with conjugate A containing the oxide It has been shown to bind to pathological tau structures in human AD brain sections. Serum was collected on day 106 after the primary immunization with guanine. The upper (AD) panels show the immunization of the black-stained cats 0, 1, and 3 months before the first immunization. Shown is staining of the parietal cortex, including tau tangles, from a diV donor. The bottom (control) panel shows Staining of parietal cortex from a Braak stage 0 donor is shown. Serum was diluted 1:100. Sections were then stained with goat anti-monkey antibody at 1:100 using DAB developer. The color was visualized.

[0259] [Example 5] Liposomal vaccines with one or two adjuvants Adding two adjuvants to the improved liposomal vaccine enhances the specificity of tau phosphopeptides. The level of specific IgG antibody titers and the consistency of antibody responses between individuals are increased.

[0260] Adult rhesus macaques (n = 3 males and 3 females / group) were incubated at 1, 29, 85, and and on day 169, 1800 μg of tetrapalmitoylated phosphorylated tau acetate of SEQ ID NO: 2 Peptide / dose control liposomal vaccine or 3D-(6-acyl)PHAD (registered trademark) (Trademark) Adjuvant alone (Liposome X, Figure 7A), lipidated CpG2006 oligonucleotide Either nucleotide adjuvant alone (liposome Y, Figure 7B) or 3D-(6 -acyl) PHAD® and lipidated CpG2006 oligonucleotide azide encapsulated T50 T cells containing both the vasodilators (liposome Z, Figure 7C). The mice were subcutaneously immunized with an improved liposomal vaccine containing a vesicle epitope. Before the epidemic and 8, 22, 36, 50, 64, 78, 92, 106, 120, 134, 1 The serum specific Ig was isolated on days 48, 162, 176, and 190. The G antibody titer was measured using the phosphorylated tau peptide of SEQ ID NO: 2 as a coating antigen and the anti- The resulting antibody levels were determined by ELISA using a IgG secondary antibody. The endpoint titers (last serum dilution that induces a positive response) for each individual monkey over time were Each immunization group is represented in one panel (Fig. 7A-C). The geometric mean ± 95% confidence intervals of the endpoint titers are presented in Figure 7D. 7A-D show the results of the liposomal vaccine containing encapsulated T50 and the administration of two azidosporins. The inclusion of tau phosphopeptides increased the level and consistency of antibody responses to tau phosphopeptides. This indicates that the antibody response improved and resulted in less variability between individual monkeys. More specifically, as shown in FIG. 7D, phosphorylated tau peptide of SEQ ID NO: 2, T50 T cells epitopes (liposomes X, Y, and Z), and one or two adjuvants The improved liposomal vaccine having the T cell epitope is different from the control liposomal vaccine having no T cell epitope. The improved liposomal vaccine induced higher titers against tau phosphopeptides than the control. All monkeys were responders when injected with either liposome or the control liposome. In the case of Kuching, four out of six animals were responders.

[0261] [Example 6] The vaccine induced antibodies specific to concentrated paired helical fibrils (ePHF) Groups of rhesus macaques (n = 3 males and 3 females / group) were treated on days 1 and 29. In addition, vaccination delivers (i) T50 T cell epitopes and 3D-(6-acyl ) Improved liposome water containing PHAD® adjuvant alone (Liposome X) (ii) T50 T cell epitope and lipidated CpG2006 adjuvant (iii) improved liposomal vaccine containing T50 T cell antigen alone (liposome Y); The pitope and two adjuvants (3D-(6-acyl)PHAD® and an improved liposomal vaccine containing lipidated CpG2006, liposome Z); or (iv) Conjugate vaccine (phosphorylated tau of SEQ ID NO: 2 linked to CRM197) peptide) and alum and CpG oligonucleotide CpG2006 (Con The mice were immunized subcutaneously by co-injection with adjuvant A.

[0262] Preparations of enriched paired helical fibrils (ePHFs) were obtained postmortem from histologically confirmed AD subjects. From brain tissue, sarkosyl extraction of insoluble tau was performed as described by Greenberg and Davis. es modified method (Greenberg and Davies, 1991, Proc Natl Acad Sci USA, 87(15):582 7-31). Antibody titers specific to concentrated helical fibronectin (ePHF) were obtained using Meso The MS Discovery (MSD) platform was used for evaluation. D Streptavidin plate was coated with biotinylated anti-tau capture antibody (HT7-biotin After coating with a fluoride-based coating (ThermoScientific), The cells were incubated with ePHFs isolated from the oocytes, whereas the ePHF-specific IgG antibodies The antibody was then tested using a sulfo-tagged anti-human IgG antibody that cross-reacts with monkey IgG antibodies. More specifically, ePHFs were pre-saturated with 1% BSA and then biotin-labeled. MSD coated with HT-7 (Thermo Scientific) Gold small spot streptavidin 96-well plate (MSD) for 1 hour. After incubation, the plates were washed with PBST and serial dilutions of serum were added. The bound antibodies were detected using a sulfo-tagged anti-human IgG antibody. After the fixation step in 1% PFA, add Read Buffer T. The plates were analyzed using a Sector Imager (MSD). For each individual monkey, the dose was measured in arbitrary units per milliliter (AU / mL) per group. The ePHF-specific antibody titers at 50 days after the first immunization are shown together with the geometric mean of each antibody. vinegar.

[0263] Figure 8 shows that all vaccines induced high titers of ePHF-specific IgG antibodies. .

[0264] Similar results of high titer ePHF-specific IgG antibodies were also obtained from a rhesus monkey administered intramuscularly. This was also observed with other liposomes, such as liposome Z+, administered to monkey macaques.

[0265] [Example 7] Induced by liposomal and conjugate vaccines in rhesus monkeys The breadth of tau phosphopeptide-specific antibodies Groups of rhesus macaques (n = 3 males and 3 females / group) were treated on days 1 and 29. (i) Encapsulated T50 and two adjuvants: TLR4 ligand (3D -(6-acyl)PHAD®) and lipidated CpG2006 oligonucleotides (ii) an improved liposomal vaccine containing tide (liposome Z); Conjugate A (phosphorylated tau peptide of SEQ ID NO: 2 linked to CRM) and co-injected with alum and the CpG oligonucleotide CpG2006. The epitope recognition profile of the antibody was determined after the second immunization (day 50). ) after 3 hours, N-terminally biotinylated 8-mer peptides with one amino acid shift were The library and the sequence of the phosphorylated tau peptide of SEQ ID NO: 2 that covers it, and Sequence number 4 (VYKSPVVSGDTSPRHL, the same amino acid sequence as SEQ ID NO: 2) (non-phosphorylated tau peptide with phospho-peptide) and the corresponding biotin-labeled full-length peptide. and determined by epitope mapping ELISA.

[0266] FIG. 9 shows that monkeys immunized with Liposome Z expressed the N-terminal region of the phosphorylated peptide of SEQ ID NO:2. While the IgG antibody bound mostly to the terminal portion (Fig. 9A), the conjugate vaccine Monkeys immunized with taucin (phosphorylated tau peptide of SEQ ID NO: 2 linked to CRM) raised IgG antibodies that mostly bound to the C-terminal portion of the tau peptide of SEQ ID NO: 2 (Figure 1). 9B).

[0267] [Example 8] T cell proliferation induced by liposomal vaccines containing encapsulated T cell epitopes Increased titers of phosphopeptide-specific IgG antibodies Three groups of C57BL / 6J mice (n=10 / group) were treated on days 0 and 14 with: i) Liposome vaccine (3D-(6-acyl)PHAD (registered trademark)) containing a TLR4 agonist ii) encapsulated T cell epitopes T50 and TL Contains R4 ligand (3D-(6-acyl)PHAD®) as an adjuvant iii) liposomal vaccines that contain a soluble ... T cell epitope T57 (i.e., dipalmitoylated T50) and TLR4 ligand Liposomal formulation containing (3D-(6-acyl)PHAD®) as an adjuvant The mice were subcutaneously immunized with a liposome vaccine (liposome T). The levels of peptide-specific IgG antibodies were measured in mouse plasma 21 and 35 days after the first injection. Results are reported as individual mouse values ​​in arbitrary units (AU) The results are presented along with the geometric mean ± 95% CI per group in / mL. To achieve this, a liposomal vaccine containing encapsulated T50 (liposome S) was used. The vaccines were administered 21 days after the first immunization, followed by the control liposomal vaccine (liposome R) and a liposomal vaccine containing tethered T cell epitopes (liposomal T) The antibody titers induced were significantly higher than those of the control group (Kruskal-Wallis test: p=0.0089 for each group). and p=0.002), and 35 days after the first immunization, compared with the control liposomal vaccine. Higher antibody titers and more effective than liposomal vaccines containing tethered T cell epitopes The antibody titers were significantly higher than those of the control group (Kruskal-Wallis test: p=0.7591 and p=0.801, respectively). p=0053) (Figure 10B).

[0268] [Example 9] The liposomal vaccine induced T cell responses specific to the incorporated T cell epitopes. Three groups of C57BL / 6J mice (n = 5 / group) were treated on days 0, 14, and 28. (i) encapsulated T cell peptide T48 (T separated by a GS linker) containing the cellular epitopes PADRE, T2, T30, and T17) and TLR4 activity an improved liposomal vaccine (liposome M) having the agent as an adjuvant (MPLA); (ii) encapsulated T52 (T cell epitopes separated by a RK linker) PADRE, T2, and T30) and a TLR4 agonist (MPLA) (iii) an improved liposomal vaccine such as Bant (liposomal N), or (iv) PBS IL-4 and IFN-γ T cell counts by ELISPOT For analysis of the response, spleens were collected from mice 42 days after the first immunization. The suspension was incubated with 10 μg / mL of medium, T48, or T52 peptide for 48 hours. The plates were then blotted with biotinylated anti-mouse IL-4 or IFN-γ monoclonal antibodies. Incubated with a single antibody and streptavidin alkaline phosphatase (AP). The spots were colored by adding AP substrate. Restimulation of mouse splenocytes with the same peptide encapsulated in the capsules resulted in IL-4 (Fig. 11B) and IFN-γ spot-forming cells (Fig. 11A), whereas PB This indicates that the spleen cells of mice injected with S were not induced by the vaccine. Addition of T cell epitopes induces activation of specific T cells, leading to the generation of tau-specific B cells It has been determined that it is possible to provide further support in antibody production against the virus.

[0269] [Example 10] Liposomes containing encapsulated and tethered T cell epitopes Some Vaccines Groups of rhesus macaques (n = 6 / group) were treated on days 1, 29, 85, and 169 ( i) Encapsulated T cell epitope T50 and TLR4 ligand (MPLA) (ii) a liposomal vaccine containing as an adjuvant (liposome L); Contains T cell epitope T46 and TLR4 ligand (MPLA) as adjuvants (iii) a liposomal vaccine (liposome O), and (iv) a TLR4 ligand (MP A control liposome vaccine containing LA) as an adjuvant and no T cell epitope Bleeding was performed before immunization (day -14) and on days 8, 22, and 30. 36, 50, 64, 78, 92, 106, 120, 134, 148, 162, 176, and The antibody was isolated on days 1 and 190. The specific IgG antibody titer was determined by measuring the phosphorylation of SEQ ID NO:2. Using tau peptide as the coating antigen and an anti-monkey IgG secondary antibody, EL The resulting antibody levels were determined as endpoint titers (those inducing a positive response). The mean serum concentration was calculated as the final serum dilution (Figure 12). Data were expressed as the geometric mean per group. As shown in Figure 1, liposomal vaccines containing encapsulated T cell epitopes (liposomes) liposome L) and liposome vaccines containing tethered T cell epitopes (liposomes Each of these vaccines was higher than the control liposomal vaccine without T cell epitopes. Tau phosphopeptide-specific antibody titers were induced.

[0270] [Example 11] Antibody responses in mice induced by conjugate vaccines Female BALB / c mice (14 mice / group) were treated with either conjugate B or conjugate C. Gate C (containing SEQ ID NO: 1 or SEQ ID NO: 3 covalently linked to KLH) was used to generate the IgG1A-specific ... A potent multicomponent adjuvant (Sigma Ad Juvant System®, Sigma-Aldrich, hereafter Ribi ) or a single-component depot adjuvant (Alhydrogel® Adjuvant 2% or aluminum hydroxide gel, InvivoGen, hereafter referred to as alum The subjects were immunized with the vaccine candidate adjuvanted with either the IgG1 or IgG2 sequences (referred to as IgG1 sequences). The amino acid sequence of number 1 differs by only one amino acid compared to that of the mouse protein. While the sequence of SEQ ID NO: 3 is 100% conserved between humans and mice, Therefore, the selected epitope is reasonably assumed to be a mouse "self" protein. This makes the mouse a relevant model for investigating the limitations that immune tolerance may impose on immunogenicity. It should be Dell.

[0271] Flow cytometry is used as a primary measure of vaccine immunogenicity, and is performed at the vaccine injection site. To measure the induction of T follicular helper cells (TfH) in draining cervical lymph nodes TfH inhibits, among other molecules, CXCR5, PD -1, and ICOS expression, + T cell population TfH expands after exposure to a vaccine or other immune stimuli and acts as a parental colony for B cells in germinal centers. Supports sexual maturation (Crotty, 2011, Annual Reviews of Immunology. Vol 29:p621-663 ). The number of induced TfHs was similar to that of humans (Bentebibel et al., 2013, Sci Transl Med., 5(17) 6):176ra32, Spensieri et al., 2013, Proc Natl Acad Sci US A., 110(35):14330-5) and positively correlates with the protective efficacy of the vaccine in small animals. The vaccine and control immunization with KLH plus adjuvant resulted in a significant reduction in the number of vaccinated mice. Furthermore, the draining cervical lymph nodes were analyzed 7 days after the first immunization. When collected, active vaccine (Conjugate B and Conjugate C groups) or All animals receiving active placebo (KLH) + alum were compared with those receiving inactive placebo ( had significantly more TfH than animals given KLH-TAUVAC- P = 0.0044 for p7.1 and P = 0.048 for KLH-TAUVAC-p22.1 2, P = 0.0063 for KLH, using ANOVA test followed by Dunnett's for multiple comparisons adjustment).

[0272] Day 0 and four additional time points after immunization (days 14, 28, 56, and 84, Figure Antibodies that bind to tau phosphopeptides and KLH in ELISA was performed to determine the serum titer of the conjugate. Immunization with gate B revealed binding cavities reactive to the corresponding vaccine peptides. Infection was induced in animals immunized with conjugate B and Ribi adjuvant. At all time points measured, the binding titer to the vaccine peptide was The binding titers were significantly higher than those induced by Sebo (conjugate B+Ribi). Compared with KLH+Ribi) (P<0.001, using ANOVA test followed by multiple comparisons (Tukey's adjustment for variance). In the alum-adjuvanted group, the difference was The difference was significant only in the 2 groups (P = 0.001 and 0.012, respectively).

[0273] The tau-specific antibody response to conjugate C (Figure 13D) was significantly higher than that to conjugate B. The responses were generally lower in magnitude than those observed in the control group, but this was due to the different assays (different coatings). The use of different peptides precludes direct statistical comparisons between the two vaccines. Nevertheless, at 28 and 84 days after immunization (P = 0.001 and 0. 008) The antibody titer against conjugate C was measured using conjugate C + Ribi as a vaccine. In mice receiving KLH Ribi, the active compound significantly increased the risk of HIV infection compared with mice receiving the placebo KLH Ribi. The titers in the alum-adjuvanted group were not significantly different from those in the active placebo group. There wasn't.

[0274] Carrier proteins protect phosphopeptides to some extent from degradation in vivo, In vivo phosphatase digestion of peptide antigens produces some non-phosphorylated peptides. It was highly likely that this exposure would expose the immune system to conjugate B and This resulted in the purification of antibodies capable of binding to the non-phosphorylated peptide in Conjugate C. To determine whether non-phosphorylated peptides were used as coating antigens, ISA was performed. As shown in Figures 13E-F, the response to non-phosphorylated tau peptide was low. The response was comparable to that of an active placebo against the same non-phosphorylated peptide. In animals immunized with Adjugate B and Ribi, Therefore, the binding titer to the phosphorylated peptide was more effective than that to the non-phosphorylated peptide. The results were significantly higher (P = 0.009 on day 14, P < 0.001 on days 28, 56, and 84). 0001, using ANOVA test). In the alum-adjuvanted group, the differences were 56 and The difference was significant only at days 1 and 84 (P = 0.0002 and 0.001, respectively). In animals immunized with Ribi adjuvant C, the response to the phosphorylated peptide was The results were higher when bant was used alone (P<0.0001 at 28 days, 56 and 8 days). P = 0.0001 on day 4).

[0275] [Example 12] Conjugate vaccine-induced antibodies are physiologically relevant for altered tau Combined with form Whether vaccine-induced antibodies can bind to physiologically relevant forms of altered tau To further determine the effect of vaccinating mice, we used post-immunization sera from vaccinated mice. Patients with Alzheimer's disease (5 AD cases), other tauopathies (3 cases of PART, FTD, PICK, and PSP) or age-matched healthy controls Postmortem human brain sections taken from one of the healthy controls (five control cases, controls) were stained. As expected, serum from control animals (PBS and active placebo groups) did not bind to brain sections. On the other hand, it was found to bind to pTau [pSer202, pThr205] obtained from a murine clone. AT8, a monoclonal antibody that binds to tau pathology, detected tau pathology in adjacent tissue sections of the corresponding area. The active vaccine, conjugate B, and conjugate C showed strong immune reactivity (Figure 14). Serum from animals immunized with Adjuvant C not only stimulated AD sections (data not shown). ), and also bound to pathological tau structures from other tauopathies (Figure 14). Conjugate B-induced antibodies inhibited (pre)tangles, neuropil threads, and These post-immunization sera reacted with neuritic plaques. Fiber tangles and neuropil threads, neuronal inclusions in FTD-tau (MAPT P301S) tissue The fibroblasts and neuropil threads, inclusion bodies and stellate cells in some cases of Pick's disease, and the fibroblasts and neuropil threads typical of PSP It also immunoreacted with typical neural inclusions, neuropil threads, and stellate cells. Polyclonal sera induced by Conjugate C showed specific responses to each tauopathy. It also reacted with pathological tau structures. In AD cases, staining was mainly concentrated in neurofibrillary tangles. and to a lesser extent concentrated in neuritic plaques and neuropil threads. Similar results were observed at lower magnifications (data not shown).

[0276] [Example 13] Vaccine-induced antibodies are functional in mice The protective efficacy of the conjugate B vaccine was tested in an injection model of tauopathy (Peer aer et al., 2015, Neurobiol Dis., 73:83-95). In this model, gene mutations (P Mice predisposed to tauopathy by 301L showed the time course shown in Figure 15A. Following this, the mice were injected intracerebrally with concentrated PHFs isolated from human AD brains. This injection, given before the onset of underlying tauopathy, prevented the progression of tauopathy in these animals. Conversely, suppressing ePHF "seeds" by tau seeding pathways such as AT8 may accelerate tau development. When premixed with antibodies capable of inhibiting tauopathy, the induction of tauopathy is reduced (unpublished data, not shown). (Not at all).

[0277] According to the scheme in Figure 15A, we have conjugate B, Ribi, or Premixed with purified IgG from serum of animals immunized with the sex control KLH Ribi The development of tauopathy was assessed after stereotactic injection of concentrated human PHFs. Two months after injection, The brains of these mice were collected and analyzed using standard biochemical analysis in whole and sarkosyl-insoluble fractions. The amount of aggregated tau in the active fraction was determined. When ePHFs premixed with IgG from mice vaccinated with B were injected, the total In both the sarkosyl-insoluble (Fig. 15B) and sarkosyl-insoluble (Fig. 15C) fractions, the control injections The animals showed significantly lower levels of aggregated phospho-tau than those receiving thiamin monophosphate (p<0.000). 1 KLH Ribi vs. KLH-TAUVAC-p7.1 Ribi, ANOVA test (Used, followed by Holm-Bonferroni adjustment for multiple comparisons). Sarkosyl-insoluble tau is good has been accepted by the Japanese Society of Clinical Oncology and correlates with the pathological features of tauopathy. Antibodies induced by vaccination with VAC-p7.1 are protective in vivo This has proven to be effective.

[0278] [Example 14] Vaccine-induced antibodies are functional in non-human primates Rhesus macaques were adjuvanted with alum and CpG oligonucleotides. Conjugate B (n=6) or KLH (n=2) were used to detect 1, 29, 85, and Blood was collected every 14 days and immunized with conjugate B. Sera from the animals were analyzed for reactivity to the immunizing peptide using ELISA ( 16A) and human ePHF using MSD (Fig. 16B). Immunization with Jugate B resulted in sustained and consistent antibody responses to the vaccine phosphopeptide. Furthermore, all animals produced measurable antibody levels against human ePHF. bell, and three of the six animals showed high reactivity to this antigen. Sera collected from animals 50 days after primary immunization were compared with human serum from healthy individuals or AD patients. The post-immune serum from conjugate B was applied to brain sections (Fig. 16C). Pathological tau structures, i.e., neurofibrillary tangles, neuropil threads, and neuritic plaques whereas serum from mice immunized with KLH did not show any reactivity. No staining was observed in control tissues. When tested in a tau immunodepletion assay, Animals receiving conjugate B develop antibodies that can bind to and deplete tau species. While at 50 days (p=0.03, using ANOVA test followed by multiple comparisons) Dunnett's preparation for immunization), immunization with KLH did not elicit such antibodies (Figure 16D). Pre- and post-immunization sera were also used as serially diluted individual samples in neutralization assays. Change from baseline (CFB) was also measured (Figure 16E). at day 14 (baseline) and days 50, 106, and 190 post-vaccination, respectively The difference in FRET counts between the specific vaccination and the control was calculated. Response at a later date (day i ) was calculated as follows:

[0279]

number

[0280] A general linear mixed model for the above responses, with animal as a random effect, was used to model categorical responses. The vaccine group, day, and serum level variables and all their phases were treated as Lee variables. Given the exploratory nature of this study, multiple testing adjustments were not considered. Hypotheses were tested at the 5% significance level.

[0281] [Example 15] Conjugates combined with alum and CpG oligonucleotide adjuvants Mice immunized with the vaccine elicited higher titer antibody responses against the vaccine peptide. brought about Adult female C57BL / 6 mice (n = 5–6 per group) were treated with either 2 μg (Figure 17A) or 0 μg. Mice were immunized intramuscularly with 2 μg of either conjugate A vaccine (Figure 17B). The conjugate vaccine was administered alone (without adjuvant) and with alum hydroxide. with CpG oligonucleotides or with alum and CpG oligonucleotides All mice received a primary immunization on day 0 of the study. followed by a single booster immunization on day 28. Alum adjuvant dose is 500ug / mouse / injection, and the dose of CpG oligonucleotide adjuvant is 2 The graph in Figure 17 shows the results before immunization (day 0) and after the vaccine. Two time points (28 and 4) after immunization with peptide T3.5 as the coating antigen were The results of binding ELISA using serum collected from mice on day 2 are shown. The mean T3.5-specific endpoint titers are plotted, and error bars represent standard errors. The table compares antibody titers using the non-parametric Kruskal-Wallis test and shows the Pairwise group comparisons were performed using the Wilcoxon signed-rank test as a post hoc Skull-Wallis test. A statistical analysis of the evaluated results is presented.

[0282] The results, shown in Figure 17, demonstrate that at both doses, the unadjuvanted vaccine elicited a strong immune response. This example shows that alum or CpG oligonucleotides were unable to induce Or the use of a combination of both improved the magnitude of the antibody response (p≦0.0152). In animals immunized with 2 μg of vaccine, the adjuvant combination was The antibody titer was significantly higher than that of the single adjuvant (p=0.0028). In animals immunized with 0.2 μg of vaccine at 1000 rpm, the alum-CpG oligonucleotide The combination of oligonucleotides performed better than the CpG oligonucleotides alone (p=0. These data demonstrate the efficacy of alum and CpG oligonucleotide adjuvants. It supports the use of combinations.

[0283] [Example 16] Liposomal vaccines with various tau peptide:T cell epitope ratios have demonstrated high and sustained efficacy. Induce sustained levels of tau phosphopeptide-specific IgG antibody titers Adult rhesus macaques (n = 6 / group) were treated on days 1, 29, 85, and 169. 3D-(6-Acyl) PHAD® and lipidated CpG2006 oligonucleotides Both contained 400 μg / mL of phosphorylated tagetes of SEQ ID NO: 2. u) peptide and 100ug / mL of T50 (Liposome Z), ii) 1200ug / mL L of phosphorylated tau peptide of SEQ ID NO: 2 and 1200 μg / mL of T50 (liposomes Z + ), iii) 400 μg / mL of phosphorylated tau peptide of SEQ ID NO: 2 and 400 μg / mL of g / mL T50 (liposome Z ++ ), iv) 1200ug / mL of phosphorus of SEQ ID NO:2 Oxidized tau peptide and 300 μg / mL of T50 (liposome Z +++ ) 180 in an improved liposomal vaccine with capsule-encapsulated T50 T cell epitope 0 μg of acetate tetrapalmitoylated phosphorylated tau peptide of SEQ ID NO: 2 / dose Bleeding was performed before immunization and at 8, 22, 36, 50, 64, 78, and 92 minutes after immunization. , 106, 120, 134, 148, 162, 176, and 190 days, and antibodies were The specific IgG antibody titer in the serum was measured by coating the phosphorylated tau peptide of SEQ ID NO: 2. as the target antigen and an anti-monkey IgG secondary antibody. The resulting antibody levels were measured as endpoint titers (positive responses) for each individual monkey over time. The number of endpoint titers per group was calculated as the final serum dilution that elicited a response. The mean ± 95% confidence intervals are presented in Figure 18 and represent the mean values ​​for all four liposomes tested. The vaccine induced high and sustained titers against tau phosphopeptides. There are.

[0284] SEQ ID NO:1 - Phosphotau peptide (7.1) GDRSGYS[pS]PG[pS]PG[pT]PGRSRSRT SEQ ID NO:2 - Phosphotau peptide (T3.5) VYK[pS]PVVSGDT[pS]PRHL SEQ ID NO:3 - Phosphotau peptide (22.1) SSTGSIDMVD[pS]PQLA[pT]LA SEQ ID NO: 4 - Tau peptide VYKSPVVSGDTSPRHL SEQ ID NO:5 - Phosphotau peptide RENAKAKTDHGAEIVYK[pS]PVVSGDT[pS]PRHL SEQ ID NO:6 - Phosphotau peptide RQEFEVMEDHAGT[pY]GL SEQ ID NO:7 - Phosphotau peptide PGSRSR[pT]P[pS]LPTPPTR SEQ ID NO:8 - Phosphotau peptide GYSSPG[pS]PG[pT]PGSRSR SEQ ID NO:9 - Phosphotau peptide GDT[pS]PRHL[pS]NVSSTGSID SEQ ID NO: 10 - Phosphotau peptide PG[pS]PG[pT]PGSRSR[pT]P[pS]LP SEQ ID NO:11 - Phosphotau peptide HL[pS]NVSSTGSID SEQ ID NO: 12 - Phosphotau peptide VSGDT[pS]PRHL SEQ ID NO: 13-T50 T cell epitope AKFVAAWTLKAAAVVRQYIKANSKFIGITELVVRFNNFTV SFWLRVPKVSASHLE-NH2 SEQ ID NO: 14-T46 T cell epitope AKFVAAWTLKAAAGSQYIKANSKFIGITELGSFNNFTVSF WLRVPKVSASHLEK(Pal)K(Pal)-NH2 SEQ ID NO: 15 - T48 helper T cell epitope AKFVAAWTLKAAAGSQYIKANSKFIGITELGSFNNFTVSF WLRVPKVSASHLEGSLINSTKIYSYFPSVISKVNQ-NH2 SEQ ID NO: 16 - T51 helper T cell epitope AKFVAAWTLKAAARRQYIKANSKFIGITELRRFNNFTVSF WLRVPKVSASHLE-NH2 SEQ ID NO: 17 - T52 helper T cell epitope AKFVAAWTLKAAAARKQYIKANSKFIGITELRKFNNFTVSF WLRVPKVSASHLE-NH2 SEQ ID NO: 18 - CpG2006 (also known as CpG7909) 5'-tcgtcgttttgtcgttttgtcgtt-3' where lowercase letters indicate phosphorothioate (ps) internucleotide linkages. SEQ ID NO: 19-CpG1018 5'-tgactgtgaacgttcgagatga-3' where lowercase letters indicate phosphorothioate internucleotide linkages SEQ ID NO: 20-CpG2395 5'-tcgtcgttttcggcgcgcgccg-3' where lowercase letters indicate phosphorothioate internucleotide linkages SEQ ID NO: 21-CpG2216 5'-ggGGGACGATCGTCgggggg-3' where lowercase letters indicate phosphorothioate internucleotide linkages and uppercase letters indicate phosphodiester means ste(po) bond SEQ ID NO: 22-CpG2336 5'-gggGACGACGTCGTGgggggg -3', where lowercase letters indicate phosphorothioate internucleotide linkages and uppercase letters indicate phosphodiester means stell coupling SEQ ID NO: 23 - Pan DR epitope (PADRE) peptide AKFVAAWTLKAAA SEQ ID NO:24-P2 QYIKANSKFIGITEL SEQ ID NO:25-P30 FNNFTVSFWLRVPKVSASHLE SEQ ID NO: 26-TT 586~605 LINSTKIYSYFPSVISKVNQ SEQ ID NO:27 - Palmitoylated phosphotau peptide (palmitoylated 7.1) K(pal)K(pal)GDRSGYS[pS]PG[pS]PG[pT]PGSRS RTK(pal)K(pal) SEQ ID NO:28 - Palmitoylated phosphotau peptide (T3, palmitoylated T3.5) K(pal)K(pal)VYK[pS]PVVSGDT[pS]PRHLK(pal) K(pal) SEQ ID NO: 29 - Palmitoylated phosphotau peptide (palmitoylated 22.1) K(pal)K(pal)SSTGSIDMVD[pS]PQLA[pT]LAK(pa l)K(pal) SEQ ID NO:30 - Palmitoylated tau peptide K(pal)K(pal)VYKSPVVSGDTSPRHLK(pal)K(pal) SEQ ID NO:31 - Palmitoylated phosphotau peptide K(pal)K(pal)RENAKAKTDHGAEIVYK[pS]PVVSGDT [pS]PRHLK(pal)K(pal) SEQ ID NO:32 - Palmitoylated phosphotau peptide K(pal)K(pal)RQEFEVMEDHAGT[pY]GLK(pal)K(p al) SEQ ID NO: 33 - Palmitoylated phosphotau peptide K(pal)K(pal)PGSRSR[pT]P[pS]LPTPPTRK(pal) K(pal) SEQ ID NO: 34 - Palmitoylated phosphotau peptide K(pal)K(pal)GYSSPG[pS]PG[pT]PGSRSRK(pal) K(pal) SEQ ID NO: 35 - Palmitoylated phosphotau peptide K(pal)K(pal)GDT[pS]PRHL[pS]NVSSTGSIDK(pa l)K(pal) SEQ ID NO: 36 - Palmitoylated phosphotau peptide K(pal)K(pal)PG[pS]PG[pT]PGSRSR[pT]P[pS]L PK(pal)K(pal) SEQ ID NO: 37 - Palmitoylated phosphotau peptide K(pal)K(pal)HL[pS]NVSSTGSIDK(pal)K(pal) SEQ ID NO: 38 - Palmitoylated phosphotau peptide K(pal)K(pal)VSGDT[pS]PRHLK(pal)K(pal) SEQ ID NO:39 - T50 without C-terminal amide AKFVAAWTLKAAAVVRQYIKANSKFIGITELVVRFNNFTV SFWLRVPKVSASHLE SEQ ID NO: 40 - T46 without -Lys(Pal)-Lys(Pal)-NH2 at the C-terminus AKFVAAWTLKAAAGSQYIKANSKFIGITELGSFNNFTVSF WLRVPKVSASHLE SEQ ID NO: 41 - T48 without C-terminal amide AKFVAAWTLKAAAGSQYIKANSKFIGITELGSFNNFTVSF WLRVPKVSASHLEGSLINSTKIYSYFPSVISKVNQ SEQ ID NO: 42 - T51 without C-terminal amide AKFVAAWTLKAAARRQYIKANSKFIGITELRRFNNFTVSF WLRVPKVSASHLE SEQ ID NO: 43 - T52 without C-terminal amide AKFVAAWTLKAAAARKQYIKANSKFIGITELRKFNNFTVSF WLRVPKVSASHLE SEQ ID NO: 44-T57 AKFVAAWTLKAAAVVRQYIKANSKFIGITELVVRFNNFTV SFWLRVPKVSASHLE-K(Pal)K(Pal)-NH2

[0285] References [Table 1]

Claims

1. a. A tau peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5, 9, and 12, which is presented on the surface of the liposome; and b. a helper T cell epitope comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 24, 25, and 26; c. a lipidated CpG oligonucleotide, wherein the CpG oligonucleotide comprises one or more phosphorothioate internucleotide linkages, and the CpG oligonucleotide is covalently attached to at least one cholesterol via a linker; d. Monophosphoryl lipid A (MPLA) and A liposome comprising:

2. The liposome of claim 1, further comprising one or more lipids selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoryl-3'-rac-glycerol (DMPG), and cholesterol. Claim 3: a. the tau peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 31, 35, and 38; b. the lipidated CpG oligonucleotide has a nucleotide sequence selected from the group consisting of SEQ ID NO:18 to SEQ ID NO:22; c. the helper T cell epitope comprises the amino acid sequence of SEQ ID NO:23, the amino acid sequence of SEQ ID NO:24, and the amino acid sequence of SEQ ID NO:25, wherein the amino acid sequences are covalently linked to each other, optionally via one or more linkers; The liposome of claim 1.

4. The liposome described in claim 3, wherein the helper T cell epitope comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 40, 41, 42 and 43.

5. The liposome described in claim 3, wherein the helper T cell epitope comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 17 and 44.

6. a. A tau peptide comprising the amino acid sequence of SEQ ID NO: 2, wherein the tau peptide is presented on the surface of the liposome; b. a helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 40, 41, 42, and 43; c. a lipidated CpG oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO:18-SEQ ID NO:22, wherein the CpG oligonucleotide is covalently attached to at least one cholesterol via a linker; and d. Monophosphoryl lipid A (MPLA) and A liposome comprising:

7. The liposome described in claim 6, further comprising one or more lipids selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoryl-3'-rac-glycerol (DMPG), and cholesterol.

8. The liposome described in claim 6, wherein the helper T cell epitope has the amino acid sequence of SEQ ID NO:

39.

9. The liposome described in claim 6, wherein the helper T cell epitope has the amino acid sequence of SEQ ID NO:

40.

10. The liposome described in claim 6, wherein the helper T cell epitope has the amino acid sequence of SEQ ID NO:

41.

11. The liposome described in claim 6, wherein the helper T cell epitope has the amino acid sequence of SEQ ID NO:

42.

12. The liposome described in claim 6, wherein the helper T cell epitope has the amino acid sequence of SEQ ID NO:

43.

13. The liposome described in claim 6, wherein the tau peptide has the amino acid sequence of SEQ ID NO:

28.

14. a. A tau peptide comprising the amino acid sequence of SEQ ID NO: 28, wherein the tau peptide is presented on the surface of the liposome; and b. a helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, and 15; c. a lipidated CpG oligonucleotide having the nucleotide sequence of SEQ ID NO: 18, wherein said CpG oligonucleotide is covalently attached to at least one cholesterol via a linker; d. Monophosphoryl lipid A (MPLA) and A liposome comprising:

15. The liposome of claim 14, further comprising one or more lipids selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoryl-3'-rac-glycerol (DMPG), and cholesterol.

16. The liposome described in claim 14, wherein the helper T cell epitope has the amino acid sequence of SEQ ID NO:

13.

17. The liposome described in claim 14, wherein the helper T cell epitope has the amino acid sequence of SEQ ID NO:

14.

18. The liposome described in claim 14, wherein the helper T cell epitope has the amino acid sequence of SEQ ID NO:

15.

19. A pharmaceutical composition comprising a liposome according to any one of claims 1 to 18 and a pharmaceutically acceptable carrier.

20. A pharmaceutical composition as described in claim 19 for inducing an immune response in a subject suffering from a neurodegenerative disorder, wherein the neurodegenerative disease or disorder is caused by or associated with the formation of neurofibrillary lesions.

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